Molecules that are vitamin d receptor agonists and histone deacetylase inhibitors

Abstract

There is provided a compound of formula I or a pharmaceutically acceptable salt or solvate thereof, (Formula I) wherein W is a 5 or 6 membered heteroaryl group comprising at least one nitrogen atom; X1 is CO or O; X2, is NH or CH2; Y is O or CH2, and n is an integer of from 2 to 5. The compound is particularly useful for the treatment of proliferative diseases such as psoriasis or a cancer selected from the group consisting of leukemia, non-small cell lung cancer, colon cancer, central nervous system cancer, melanoma, ovarian cancer, renal cancer, prostate cancer, and breast cancer.

Description

MOLECULES THAT ARE VITAMIN D RECEPTOR AGONISTS AND HISTONE DEACETYLASE INHIBITORSTECHNICAL FIELD

[0001] This disclosure relates to the field of small molecules that are vitamin D receptor agonists and also histone deacetylate inhibitors.BACKGROUND OF THE ART

[0002] Although initially identified as a cure for nutritional rickets, a disease of bone growth, vitamin D has attracted extensive interest because of its “non-classical” actions. Apart from nutritional supplementation, vitamin D is obtained from limited dietary sources or from cutaneous exposure to adequate solar ultraviolet B (UVB) irradiation, which induces the photochemical and thermal conversion of cholesterol precursor, 7-dehydrocholesterol. This cleaves the steroid B ring and generates secosteroidal vitamin D3 (cholecalciferol). The active form of vitamin D, 1 ,25- dihydroxyvitamin D (1 ,25D, calcitriol) is produced via sequential hydroxylations, first at the 25 position (25- hydroxy vitamin D, calcidiol), and then in a highly regulated, tissue-specific manner at the 1a position by the enzyme CYP27B1. 1 ,25D binds to and activates the vitamin D receptor (VDR), a protein that is widely expressed, including in several tissues unrelated to calcium homeostasis. The VDR functions as a ligand-regulated transcription factor and binds to regulatory regions of genes controlling cell cycle regulation, differentiation and immune function. Notably, there is evidence from clinical data that vitamin D supplementation can be cancer-preventive.Compound 1

[0003] 1 ,25D analogs have been investigated for their potential in treatment of proliferative disorders, such as cancer and psoriasis. Indeed, 1 ,25D and its analogs are antiproliferative in several cancer models, including breast cancer. A secosteroidal 1 ,25D analogue, EB1089, was efficaciousin a head and neck squamous carcinoma (HNSCC) model. Unfortunately, typical of other cancers, there are also multiple HNSCC models that are resistant to 1 ,25D. Consequently, 1 ,25D analogues have generally failed as monotherapies because of acquired tumor resistance. Importantly, however, vitamin D signaling is often retained in resistant cells, underlying the potential for 1 ,25D and its analogs in combination therapies. 1 ,25D and histone deacetylase inhibitors (HDACis) act synergistically in 1 ,25D-resistant models. In addition, VDR signaling and the HDACi sodium butyrate cooperate to induce colon cancer cell differentiation. In the nucleus, histone deacetylases (HDACs) control acetylation of histones, transcription factors and cofactors, and thus regulate gene transcription. However, they also control acetylation of other non-nuclear proteins such as tubulin and HSP90. Prototypical HDACi trichostatin A (TSA), initially isolated for its antifungal activity, also has potent antiproliferative properties. Class I, II and IV HDACs are zinc metalloenzymes, and TSA is typical of HDACi in that it is composed of a zinc-chelating group (frequently a hydroxamic acid) flanked by a linker and a cap group, which can be highly heterogeneous in structure. Several HDACi, including SAHA (vorinostat), have been approved for clinical use for cutaneous and peripheral T-cell lymphoma, and multiple myeloma, or are in clinical trials.

[0004] Fully integrated bifunctional hybrid molecules that combine VDR agonism with HDACi activity within the backbone of a VDR agonist have been developed. They thus represent a form of combination therapy, which is now the norm in the treatment of cancer, as they regulate distinct but biochemically complementary targets. Initially, triciferol was developed and other secosteroidal hybrids by replacing the cholesterol-like 1 ,25D sidechain of 1 ,25D with the zinc-chelating dienylhydroxamic acid unit of TSA. The hydroxamic acid successfully mimics the 1 ,25D 25-OH group, while the secosteroid functions as an HDACi cap group. As synthesis of secosteroidal hybrids required numerous (>25) steps, HDACi activity was then incorporated into the more easily assembled diarylpentane 1 ,25D analogue LG-190,178, resulting in hybrid JF-B01. Receptor binding of JF-B01 and its analogs was confirmed by fluorescence polarization assays and crystal structures of non- secosteroidal hybrid / VDR complexes. Subsequently, the HDACi potency was improved by removal of the methyl group in the aromatic ring adjacent to the HDACi group and lengthening the side chain (see DK-366, DK-367 and DK-406).(LG-190, 178) Compound 2(triciferol) Compound 5(JF-B01)Compound 6a(DK-406) Compound 6d

[0005] The compounds were bioavailable and inhibited tumor growth in the mouse 4T1 triplenegative breast cancer (TNBC) model, under conditions where 1 ,25D and SAHA given alone or in combination were not efficacious. 4T 1 cells are derived from a TNBC malignancy in Balb / c mice, form rapidly growing primary tumors and are aggressively metastatic. TNBC, by definition, lacks the markers of other forms of breast cancer (estrogen and progesterone receptors or amplified epidermal growth factor receptor HER2), and represents about 15-20% of all breast malignancies. While results with the 4T 1 model were encouraging, the hydrophobic diarylpentane core made solubilizing DK-367 and -406 problematic. One of the benzene rings was replaced by a pyridine (AM-193), which did notsubstantially compromise bifunctionality. However, gains in solubility of AM-193 relative to DK-367, its closest structural analogue, were relatively modest, and efficacy of AM-193 in vivo is limited by its relative toxicity.

[0006] Improvements in the solubility of the compounds to improve bioavailability and efficacy, as well as a reduction of the toxicity is therefore desired.SUMMARY

[0007] In one aspect, there is provided a compound of formula I or a pharmaceutically acceptable salt or solvate thereofW is a 5 or 6 membered heteroaryl group comprising at least one nitrogen atom as a heteroatom in the heteroaryl group, and the heteroaryl group has a methyl substitution, when possible, adjacent to Y;Xi is CO or O,X2 is NH or CH2, wherein when Xi is CO, X2 is NH and when Xi is O, X2 is CH2;Y is O or CH2;n is an integer of from 2 to 5; and when Xi is CO, W is a 6 membered heteroaryl group with only one nitrogen atom or is a 5 membered heteroaryl group. when Xi is O, W is a 5 membered heteroaryl group and when Xi is CO, W is a 6 membered heteroaryl group with only one nitrogen atom or is a 5 membered heteroaryl group.

[0008] In some embodiments, n is an integer of from 2 to 4 when Xi is O and n is an integer of from 3 to 5 when Xi is CO.

[0009] In some embodiments, W is selected from the group consisting of

[0011] Preferably,X1is O.

[0012] In some embodiments, the compound is

[0013] The present compound is useful in the treatment of a cancer selected from the group consisting of leukemia, non-small cell lung cancer, colon cancer, central nervous system cancer, melanoma, ovaria cancer, renal cancer, prostate cancer, and breast cancer, or for the treatment of psoriasis. Accordingly, there is also provided a pharmaceutical composition comprising the compound and a pharmaceutically acceptable excipient for these treatments.

[0014] In one aspect, there is provided a method of treating a patient with a cancer selected from the group consisting of leukemia, non-small cell lung cancer, colon cancer, central nervous system cancer, melanoma, ovaria cancer, renal cancer, prostate cancer, and breast cancer, comprising administering to the patient the compound of the present disclosure or the pharmaceutical composition comprising same. Optionally, an immune checkpoint blockade agent is administered to the patient.

[0015] In still a further aspect, there is provided a method of treating a patient with psoriasis, comprising administering to the patient the compound of the present disclosure or the pharmaceutical composition comprising same.

[0016] Many further features and combinations thereof concerning the present improvements will appear to those skilled in the art following a reading of the instant disclosure.DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1A is a bar graph showing the Cyp24a1 induction by ZG compounds, along with DK- 406, AM-193, 1,25D and SAHA compounds in 4T1 cells.

[0018] FIG. 1 B is a bar graph showing the Cyp24a1 induction by ZG compounds, along with DK- 406, AM-193, 1,25D and SAHA compounds in 4TO7 cells.

[0019] FIG. 2A is a bar graph showing the cytotoxicity of different compounds in mouse 4T 1 cells.

[0020] FIG. 2B is a bar graph showing the cytotoxicity of different compounds in the human TNBC cell line MDA-MB-231.

[0021] FIG. 3A is a bar graph showing the quantification of triplicate biological replicates of different compounds on hyperacetylation of tubulin in 4T 1 cells.

[0022] FIG. 3B is a bar graph showing the quantification of triplicate biological replicates of different compounds on hyperacetylation of H3K9 in 4T1 cells.

[0023] FIG. 3C is a bar graph showing the quantification of triplicate biological replicates of different compounds on hyperacetylation of H3K27 in 4T 1 cells.

[0024] FIG. 3D is a bar graph showing the quantification of triplicate biological replicates of different compounds on hyperacetylation of tubulin in 4TO7 cells.

[0025] FIG. 3E is a bar graph showing the quantification of triplicate biological replicates of different compounds on hyperacetylation of H3K9 in 4TO7 cells.

[0026] FIG. 3F is a bar graph showing the quantification of triplicate biological replicates of different compounds on hyperacetylation of H3K27 in 4TO7 cells.

[0027] FIG. 4A is a graph showing the dose response curve of HDAC1 by ZG-126 compound.

[0028] FIG. 4B is a graph showing the dose response curve of HDAC2 by ZG-126 compound.

[0029] FIG. 4C is a graph showing the dose response curve of HDAC3 by ZG-126 compound.

[0030] FIG. 4D is a graph showing the dose response curve of HDAC6 by ZG-126 compound.

[0031] FIG. 5A is a graph showing the dose response curve of HDAC4 by ZG-126 compound.

[0032] FIG. 5B is a graph showing the dose response curve of HDAC5 by ZG-126 compound.

[0033] FIG. 5C is a graph showing the dose response curve of HDAC7 by ZG-126 compound.

[0034] FIG. 5D is a graph showing the dose response curve of HDAC8 by ZG-126 compound.

[0035] FIG. 5E is a graph showing the dose response curve of HDAC9 by ZG-126 compound.

[0036] FIG. 5F is a graph showing the dose response curve of HDAC10 by ZG-126 compound.

[0037] FIG. 5G is a graph showing the dose response curve of HDAC11 by ZG-126 compound.

[0038] FIG. 6A is a bar graph showing the Cyp24a1 induction by ZG126, DK406, AM193 along with 1 ,25D and SAHA as controls in B16-F10 cells.

[0039] FIG. 6B is a Western blot showing the effects of 1,25D, SAHA and compounds on hyperacetylation of tubulin, H3K9 and H3K27 in B16-F10 cells.

[0040] FIG. 6C is a bar graph showing a quantification of hyperacetylation in B16-F10 cells of tubulin.

[0041] FIG. 6D is a bar graph showing a quantification of hyperacetylation in B16-F10 cells of H3K9.

[0042] FIG. 6E is a bar graph showing a quantification of hyperacetylation in B16-F10 cells of H3K27.

[0043] FIG. 6F is a bar graph showing the assessment of cytotoxicity in B16-F10 cells by Gio™ MT assay.

[0044] FIG. 7A is a bar graph showing Cyp24a1 induction in B16-F10 tumors of B16-F10 melanoma model treated with 2 doses 24 hrs apart of 5 mg / kg ZG-126.

[0045] Fig. 7Ai is western blot showing the effect ZG126 functioning as an HDACi at well- tolerated therapeutic doses in vivo in B16-F10 tumors.

[0046] FIG. 7B is a bar graph showing the body weights of B16-F10 melanoma model of Fig. 7A after 28 days.

[0047] FIG. 7C is a bar graph showing the effects of 1,25D 0.25 mg / kg, SAHA (80 mg / kg), AM- 193 (0.2 mg / kg), ZG-126 (5 mg / kg) or gemcitabine (50 mg / kg) on B16 tumor growth.

[0048] FIG. 7D is a graph showing endpoint tumor sizes from the experiment of Fig. 7C.

[0049] FIG. 8A is a bar graph showing the weight loss in the 4T1 TNBC model after ZG-126 treatment.

[0050] FIG. 8B is a photograph showing primary 4T 1 tumors from control and treated mice.

[0051] FIG. 8C is a graph showing the effects over time on 4T1 tumor growth in Balb / c mice based on different treatments.

[0052] FIG. 8D is a photograph showing the effects on 4T1 tumor growth in Balb / c mice based on different treatments.

[0053] FIG. 9A is a graph showing gene expression profile of Ccl2 expressed in 4T 1 cells based on different in vitro treatments.

[0054] FIG. 9B is a bar graph showing the gene expression of Ccl5 expressed in 4T 1 cells based on different in vitro treatments.

[0055] FIG. 9C is a bar graph showing gene expression of Ccl20 expressed in 4T 1 cells based on different in vitro treatments.

[0056] FIG. 9D is a bar graph showing gene expression of Cxcl10 expressed in 4T1 cells based on different in vitro treatments.

[0057] FIG. 9E is a bar graph showing gene expression of IL1 A expressed in 4T 1 cells based on different in vitro treatments.

[0058] FIG. 9F is a bar graph showing the density of macrophages as judged by F4 / 80 staining.

[0059] FIG. 9G is a bar graph showing the density of macrophages as judged by the ratio of Arg 1- positive macrophages to total macrophages.

[0060] FIG. 9H is a microscopy image showing the immunofluorescence of 4T1 tumors stained for macrophage marker, F4 / 80 and M2 macrophage-specific marker, Arg1 , these tumors are from animals treated with vehicles.

[0061] FIG. 9I is a microscopy image showing the immunofluorescence of 4T 1 tumors stained for macrophage marker, F4 / 80 and M2 macrophage-specific marker, Arg1, these tumors are from animals treated with ZG-126.

[0062] FIG. 10A is a microscopy image of an animal tumor treated with vehicle and stained for F4 / 80.

[0063] FIG. 10B is a microscopy image of an animal tumor treated with 1,25D (0.25 pg / kg) and stained for F4 / 80.

[0064] FIG. 10C is a microscopy image of an animal tumor treated with SAHA (80 mg / kg) and stained for F4 / 80.

[0065] FIG. 10D is a microscopy image of an animal tumor treated with 1,25D and SAHA and stained for F4 / 80.

[0066] FIG. 10E is a microscopy image of an animal tumor treated with ZG-126 and stained for F4 / 80.

[0067] FIG. 10F is a microscopy image of an animal tumor treated with vehicle and stained for Arginase 1 (Arg1).

[0068] FIG. 10G is a microscopy image of an animal tumor treated with 1,25D (0.25 pg / kg) and stained for Arg1.

[0069] FIG. 10H is a microscopy image of an animal tumor treated with SAHA (80 mg / kg) and stained for Arg1.

[0070] FIG. 101 is a microscopy image of an animal tumor treated with 1 ,25D and SAHA and stained for Arg1.

[0071] FIG. 10J is a microscopy image of an animal tumor treated with ZG-126 and stained for Arg1.

[0072] FIG. 10K is a microscopy image combining the staining of Figs. 10A and 10F.

[0073] FIG. 10L is a microscopy image combining the staining of Figs. 10B and 10G.

[0074] FIG. 10M is a microscopy image combining the staining of Figs. 10C and 10H.

[0075] FIG. 10N is a microscopy image combining the staining of Figs. 10D and 101.

[0076] FIG. 10O is a microscopy image combining the staining of Figs. 10E and 10J.

[0077] FIG. 11A is a bar graph showing the induction of VDR target gene Cyp21A1 in B16-F10 melanoma cells based on treatment with different compounds.

[0078] FIG. 11B is a western blot showing the effect of different compounds on tubulin, H3K9, and H3K27 in B16-F10 melanoma cells.

[0079] FIG. 11 C is a bar graph showing the quantification of the western blot (Fig. 11 B) of tubulin.

[0080] FIG. 11 D is a bar graph showing the quantification of the western blot (Fig. 11 B) of H3K9.

[0081] FIG. 11 E is a bar graph showing the quantification of the western blot (Fig. 11 B) of H3K27.

[0082] FIG. 12A is a bar graph showing the induction of VDR target gene Cyp21A1 in humanA375 melanoma cells based on treatment with different compounds.

[0083] FIG. 12B is a western blot showing the effect of different compounds on tubulin, H3K9, and H3K27 in human melanoma cells.

[0084] FIG. 12C is a bar graph showing the quantification of the western blot (Fig. 12B) of tubulin.

[0085] FIG. 12D is a bar graph showing the quantification of the western blot (Fig. 12B) of H3K9.

[0086] FIG. 12E is a bar graph showing the quantification of the western blot (Fig. 12B) of H3K27.

[0087] FIG. 13A is a bar graph showing the induction of VDR target gene Cyp21A1 in SK-M EL-28 melanoma cells based on treatment with different compounds.

[0088] FIG. 13B is a western blot showing the effect of different compounds on tubulin, H3K9, and H3K27 in SK-MEL-28 melanoma cells.

[0089] FIG. 13C is a bar graph showing the quantification of the western blot (Fig. 13B) of tubulin.

[0090] FIG. 13D is a bar graph showing the quantification of the western blot (Fig. 13B) of H3K9.

[0091] FIG. 13E is a bar graph showing the quantification of the western blot (Fig. 13B) of H3K27.

[0092] FIG. 14A is a bar graph showing the cytotoxicity of various compounds on B16-F10 cells.

[0093] FIG. 14B is a bar graph showing the cytotoxicity of various compounds on A375 cells.

[0094] FIG. 14C is a bar graph showing the cytotoxicity of various compounds on SK-M EL-28 cells.

[0095] FIG. 15A is a Venn diagram showing the number of genes that are being commonly and / or uniquely regulated by AC-340, AM-193 and ZG-126.

[0096] FIG. 15B is a Venn diagram showing the number of genes that are being commonly and / or uniquely regulated by AC-340, SAHA and SAHA+1,25D.

[0097] FIG. 16A is a graph showing the change in gene expression comparing 1,25D (positive log fold change, above horizontal line) and vehicle (negative log fold change, below horizontal line).

[0098] FIG. 16B is a graph showing the change in gene expression comparing SAHA (positive log fold change, above horizontal line) and vehicle (negative log fold change, below horizontal line).

[0099] FIG. 16C is a graph showing the change in gene expression comparing 1.25D+SAHA (positive log fold change, above horizontal line) and vehicle (negative log fold change, below horizontal line).

[0100] FIG. 16D is a graph showing the change in gene expression comparing AM-193 (positive log fold change, above horizontal line) and vehicle (negative log fold change, below horizontal line).

[0101] FIG. 16E is a graph showing the change in gene expression comparing ZG-126 (positive log fold change, above horizontal line) and vehicle (negative log fold change, below horizontal line).

[0102] FIG. 16F is a graph showing the change in gene expression comparing AC-340 (positive log fold change, above horizontal line) and vehicle (negative log fold change, below horizontal line).

[0103] FIG. 16G is a plot of log fold change in function of average gene expression level for DEGs in BF16-F10 cells treated with 1,25D.

[0104] FIG. 16H is a plot of log fold change in function of average gene expression level for DEGs in BF16-F10 cells treated with SAHA.

[0105] FIG. 161 is a plot of log fold change in function of average gene expression level for DEGs in BF16-F10 cells treated with 1.25D+SAHA.

[0106] FIG. 16J is a plot of log fold change in function of average gene expression level for DEGs in BF16-F10 cells treated with AM- 193.

[0107] FIG. 16K is a plot of log fold change in function of average gene expression level for DEGs in BF16-F10 cells treated with ZG-126.

[0108] FIG. 16L is a plot of log fold change in function of average gene expression level for DEGs in BF16-F10 cells treated with AC-340.

[0109] FIG. 17A is a graph showing the comparison of relative fold inductions of the 50 most induced 1 ,25D target genes by 1,25D (left bar for each gene) or AC-340 (right bar for each gene), taken from RNA sequence data in B16-F10 cells.

[0110] FIG. 17B is a bar graph showing the induction of the Abdc2 gene based on different treatments of B16-F10 cells.

[0111] FIG. 17C is a bar graph showing the induction of the Tgm2 gene based on different treatments of B16-F10 cells.

[0112] FIG. 17D is a bar graph showing the induction of the Prelp gene based on different treatments of B16-F10 cells.

[0113] FIG. 17E is a bar graph showing the downregulation of Kit based on different treatments of B16-F10 cells.

[0114] FIG. 17F is a bar graph showing the downregulation of FoxM1 based on different treatments of B16-F10 cells.

[0115] FIG. 17G is a bar graph showing the downregulation of Exo1 based on different treatments of B16-F10 cells.

[0116] FIG. 17H is a bar graph showing the downregulation of Lpar5 based on different treatments of B16-F10 cells.

[0117] FIG. 171 is a bar graph showing the downregulation of Enpp2 based on different treatments of B16-F10 cells.

[0118] FIG. 18A is a bar graph showing the relative gene expression of cMyc based on different treatments of B16-F10 cells.

[0119] FIG. 18B is a bar graph showing the relative gene expression of Mxd1 based on different treatments of B16-F10 cells.

[0120] FIG. 18C is a bar graph showing the relative gene expression of E2F1 based on different treatments of B16-F10 cells.

[0121] FIG. 18D is a bar graph showing the relative gene expression of Melk based on different treatments of B16-F10 cells.

[0122] FIG. 18E is a graph showing the relative gene expression of Ccne2 based on different treatments of B16-F10 cells.

[0123] FIG. 18F is a graph showing the relative gene expression of Cdc20 based on different treatments of B16-F10 cells.

[0124] FIG. 18G is a graph showing the relative gene expression of Cdk2 based on different treatments of B16-F10 cells.

[0125] FIG. 19A is a bar graph showing the relative gene expression of Mitf based on different treatments of B16-F10 cells.

[0126] FIG. 19B is a bar graph showing the relative gene expression of Det based on different treatments of B16-F10 cells.

[0127] FIG. 19C is a bar graph showing the relative gene expression of Pmel based on different treatments of B16-F10 cells.

[0128] FIG. 19D is a bar graph showing the relative gene expression of Tyr based on different treatments of B16-F10 cells.

[0129] FIG. 20A is a bar graph showing the induction of expression of the MHC class I component H2-D1 based on different treatments of B16-F10 cells.

[0130] FIG. 20B is a bar graph showing the induction of expression of the MHC class I component H2-K1 based on different treatments of B16-F10 cells.

[0131] FIG. 20C is a bar graph showing the induction of expression of the MHC class I component H2-Dmb1 based on different treatments of B16-F10 cells.

[0132] FIG. 20D is a bar graph showing the induction of expression of the MHC class I component CD274 based on different treatments of B16-F10 cells.

[0133] FIG. 20E is a bar graph showing the induction of expression of the MHC class I component H2-T222 based on different treatments of B16-F10 cells.

[0134] FIG. 20F is a bar graph showing the induction of expression of the MHC class I component P2m based on different treatments of B16-F10 cells.

[0135] FIG. 20G is a bar graph showing the induction of expression of the M HC class I component Tapbp based on different treatments of B16-F10 cells.

[0136] FIG. 20H is a bar graph showing the induction of expression of the MHC class I component Pvr based on different treatments of B16-F10 cells.

[0137] FIG. 21 A is a graph showing the upregulation of MHC class I proteins for different compounds.

[0138] FIG. 21 B is a graph showing the percentage increase of the upregulation of MHC class I proteins compared to the control.

[0139] FIG. 21 C is a graph showing the upregulation of p2-macroglobulin for different compounds.

[0140] FIG. 21 D is a graph showing the percentage increase of the upregulation of p2- macroglobulin compared to the control.

[0141] FIG. 22 is a bar graph showing the Cyp24a1 induction by AC-455, AC-454 or AC-451 compounds, along with 1 ,25D and SAHA compounds in 4T1 cells after 24 h.DETAILED DESCRIPTION

[0142] There is provided a compound of formula I or a pharmaceutically acceptable salt or solvate thereof.

[0143] W is a 5 or 6 membered heteroaryl group comprising at least one nitrogen atom as a heteroatom in the heteroaryl group, and the heteroaryl group has a methyl substitution, when possible, adjacent to Y. Y is O or CH2. Xi is CO or O and X2 is NH or CH2. When Xi is CO, X2 is NH and when Xi is O, X2 is CH2. n is an integer of from 2 to 5. Preferably, n is an integer of from 2 to 4 when Xi is O and n is an integer of from 3 to 5 when Xi is CO. Moreover, when Xi is O, W is a 5 membered heteroaryl group and when Xi is CO, W is a 6 membered heteroaryl group with only one nitrogen atom or is a 5 membered heteroaryl group.

[0144] The term “heteroaryl” as used herein to define W refers to an aromatic cycle comprising at least two carbon atoms linking W to Y and to the CH (see formula I), at least one nitrogen atom and optionally additional heteroatoms selected from S, O and N.

[0145] In some embodiments, W is selected from

[0147] In some embodiments, the compound of formula I is

[0148] The present compound of formula I is a molecule that provides a combination therapy in a single molecule, which greatly simplifies dosing. They provide the dual action of being equivalent or better than the active form of vitamin D and of the food and drug administration (FDA) approved HDACi SAHA. As demonstrated in the Examples section below, the present compounds display improved efficacy relative to the active form of vitamin D, the HDACi SAHA, or combinations of thetwo together. They also display greatly superior bioavailability to SAHA as they are more efficacious at 16-100-fold lower doses.

[0149] It was surprisingly found that providing a polar amide group in the hydroxamic sidechain (i.e. embodiments in formula I where Xi is CO instead of O and thus X2 is NH and not CH2) and / or providing a more polar heteroaryl group (i.e. embodiments in formula I where W is a 5 membered heteroaryl), improved solubility and efficacy compared to AM-193. The compounds obtained are bifunctional, and are more soluble and substantially better tolerated in vivo than AM-193. The IC50S for lead compounds were determined for all 11 Class I, II and IV HDACs and it was found that the compounds induce robust VDR target gene expression in vivo. Moreover, the compounds displayed efficacious anti-tumor and anti-metastatic activity in mouse models of melanoma and TNBC, both of which are malignancies in need of efficacious therapeutics.

[0150] The present compounds are useful in the treatment of proliferative diseases, such as psoriasis and cancer. The present compounds can be used for limiting, or inhibiting the proliferation of cancer cells, or causing death of cancer cells in a patient. The present compounds are particularly useful at reducing or preventing the proliferation of, or causing cell death of 1 ,25D-resistant cell lines.

[0151] Examples of cancers that can be treated by the present compounds include, but are not limited to, multiple myeloma, tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; and other tumors, including melanoma, seminoma, tetratocarcinoma, neuroblastoma and glioma. Examples of cancers include carcinoma, including that of the bladder, breast, colon, kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid, skin, oral cavity, esophagus; hematopoietic tumors of lymphoid lineage, including acute lymphocytic leukemia, B-cell lymphoma and Burketts lymphoma; hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemias and promyelocytic leukemia, multiple myeloma; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; and other tumors, including melanoma, seminoma, tetratocarcinoma, neuroblastoma and glioma. In one embodiment, the cancer is selected from leukemia, non-small cell lung cancer, colon cancer, central nervous system cancer, melanoma, ovaria cancer, renal cancer, prostate cancer, breast cancer.

[0152] In some embodiments, the present compounds are used for the treatment of a cancerthat is leukemia, non-small cell lung cancer, colon cancer, central nervous system (CNS) cancer, melanoma, ovarian cancer, renal cancer, prostate cancer and breast cancer.

[0153] Combination therapies are the standard in most cancer treatment regimens. Common therapeutic agents that can be combined with the present compounds include but are not limited toimatinib, paclitaxel, docetaxel, cisplatin, doxorubicine, vinblastine, zoledronate and / or in conjunction with antimetastatic agents, antiangionevic agents such as avastatin, and antiapoptotic compounds such as Valcade, agents targeting synthesis of estrogens or estrogen signaling through estrogen receptors including but not limited to arimidex and tamoxifen, agents targeting biosynthesis of androgens or androgen signaling through the androgen receptor including but not limited to bicalutamide, agents targeting HER2 including but not limited to trastuzumab, agents targeting BRAF including but not limited to Vemurafenib, or agents targeting members of the MAP kinase family or their upstream or downstream effector kinases.

[0154] The present compounds can also be combined with immunotherapeutic agents such as phagocytosis checkpoint blockade agents. Indeed, the combination of the present compounds and phagocytosis checkpoint blockades would lead to a co-targeting of tumour associated macrophages (TAMs) which are an important component of the tumour microenvironment (TME). Such a combination therapy would enhance the reprogramming of TAMs toward a more tumoricidal phenotype. Accordingly, in some embodiments there is provided the combination of the present compounds with immunomodulators and / or cytokine treatments such as immune checkpoint inhibitors, for the treatment of cancer.EXAMPLE 1

[0155] Previously developed compounds DK-366, DK-367 and DK-406, with their diarylpentane cores, were bifunctional in vitro, and DK-366 and -406, further tested in vivo, were bioavailable and efficacious in reducing tumor burden and the number of metastases in the 4T1 TNBC model. However, efficacy of DK-406 was limited by its relatively narrow therapeutic window and its hydrophobic diarylpentane core limited its solubility. Pyridyl-substituted AM-193, with a sidechain ether linker, was bifunctional and modestly more soluble than its closest analogue DK-367, but still less soluble than DK-406, which bears an amide in its sidechain. Moreover, AM-193 is limited by its toxicity in vivo. To optimize solubility, the pyridine core and amide linkage were combined into a new set of compounds, ZG-102, ZG-126 and ZG-132, which differ in their HDACi sidechain lengths.

[0156] Unless otherwise stated, reactions were conducted under an argon atmosphere and glassware was oven dried prior to use. Tetrahydrofuran and diethyl ether were purified by distillation from sodium under a nitrogen atmosphere. Toluene, dichloromethane and triethylamine were purified by distillation from calcium hydride under nitrogen atmosphere. Deuterated chloroform was stored over activated 4 A molecular sieves. All commercial reagents and solvents were used as purchased without further purification. Thin-layer chromatography (TLC) was carried out on glass-backed ultrapure silica TLC plates (extra hard layer, 60 A, thickness 250 pm, saturated with F-254 indicator) purchased from Silicycle. Flash column chromatography was carried out on 230-400 mesh silica gel (Silicycle) using reagent grade solvents. Proton and carbon nuclear magnetic resonance spectra were obtained on Varian™ 500 or Bruker™ 500 and 800 MHz spectrometers. Chemical shifts (5) were internally referenced to the residual proton resonance including but not limited to CDCI3 (6 7.26 ppm), CD3OD (53.31 ppm), and (CDs^SO (52.50 ppm). Coupling constants (J) are reported in Hertz (Hz). High resolution mass spectroscopy (HRMS) results were obtained by collaborators at McGill University Department of Chemistry. Compounds 1 ,25D (BML-DM200) and SAHA (#10009929) were purchased from Enzo Life Sciences and Cayman Chemical, respectively. They were both used at a final concentration of 100 nM.

[0157] Synthesis of the ZG series (Scheme 1) was modeled on the route developed for AM compounds featuring a key aza-Achmatowicz rearrangement. The precursor for the rearrangement was prepared by addition of ethyl magnesium bromide to methyl 4-benzyoxybenzoate, in 95% yield, followed by Friedel-Crafts alkylation with phthalimide-protected furylamine 9 in the presence of BF3*OEt2to afford diarylpentane 10 in 86% yield. The phthalimde protecting group in 10 was removed,in 98% yield using hydrazine, to generate free amine 11 , which is the desired precursor for aza- Achmatowicz rearrangement. The standard Achmatowicz conditions were applied, using Br2 in 2:1 MeOH / H2O, to establish the pyridine ring of intermediate 12 in 67% yield. Subsequent O-akylation of the 3-hydroxypyridine with 1-chloropinacolone afforded 13 in 98% yield. After hydrogenolysis of the benzyl group, the resulting phenol 14 was converted to a methyl ester 16 via a sequential triflation and palladium-catalyzed carbonylation in 83% yield. Reduction of the ketone in 16 with NaBH4 followed by ester saponification afforded acid 18 in 57% over two steps. With the carboxylic acid in hand, EDOHCI catalyzed amide coupling with C4, C5, and C6 amino esters afforded penultimate compound precursors 19a-c in 89-98% yields. Finally, treatment with hydroxylamine and KOH afforded desired compounds ZG-132 (20a, n=3), ZG-126 (20b, n=4), and ZG-102 (20c, n=5) in 26- 53% yields. More details on Scheme 1 , regarding the synthesis of compounds 14-18, 19a, 19b, 19c, 20a, 20b, and 20c, is provided in Table 1.Scheme 1.Table 1. Synthesis and confirmation of the compounds

[0158] Mouse 4T1 and 4TO7 TNBC cells and B16-F10 mouse melanoma cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; 319-005-CL; WISENT Inc.) supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS; 098150; WISENT Inc.). Human TNBC cells, MDA-MB- 231 were cultured in Leibovitz’s L-15 medium (323-050-CL; WISENT Inc.) with 10% heat-inactivated FBS in the absence of CO2. Cells were then treated with DMSO (vehicle), 1 ,25D, SAHA, DK-406, AM-193 or ZG compounds (102, 126 or 132) for 6 or 24 h.

[0159] First, the change in solubility was assessed for the modification of the structures. The incorporation of amide and pyridine groups in the compounds improved the solubility of DK-367 (1.91 pg / mL) to 4.40 pg / mL (DK-406) and 2.43 pg / mL (AM-193), respectively. Combining these groups in the ZG compounds further improved solubility: 4.45 pg / mL, 9.19 pg / mL and 10.71 pg / mL respectively for ZG-102, ZG-126 and ZG-132. The bifunctionality of ZG compounds was then analyzed in a battery of assays. VDR agonism was determined by evaluating the induction of the Cyp24a1 gene. Cyp24a1 encodes the enzyme that initiates catabolic degradation of 1 ,25D in a negative-feedback loop, and its gene transcription is exquisitely sensitive to the presence of the agonist-bound VDR. Agonism was assessed initially in mouse 4T1 and in related 4TO7 TNBC cells. ZG compounds induced Cyp24a1 expression in both lines (although ZG-102 and ZG-132 did not appear to be a full agonist in 4T1 cells). Apparent potencies were lower than those of DK-406 and AM-193. However, half maximal effective concentration (EC50) values could not be determined because of toxicity of compounds at concentrations above 10 pM (Figs. 1A-1 B).

[0160] To determine the cytotoxicity of the ZG molecules, the assay was performed 24 h after treatment of 4T1 , MDA-MB-231 or B16-F10 cells according to the manufacturer’s instructions (RealTime™ Gio MT cell viability assay (MTGIo), Promega - #G9712). Luminescence was measured using a 1420 Luminescence Counter Victor Light (PerkinElmer), normalized to control, and plotted. All samples were run in triplicate.

[0161] Cytotoxicity in 4T1 cells was assessed using a RealTime-Glo™ metabolism (MT) cell viability assay, which measures the release of a modified luciferase substrate that can only be generated by viable cells. In these assays, the IC50 of ZG-126 was comparable to that of AM-193 (0.89 vs 0.74 pM; Fig. 2A, see Table 2 for all IC50 values) and was 5-50-fold lower than those of ZG- 132 and ZG-102, respectively (Fig. 2A, Table 2). ZG-126 was also more potent than DK-406 (0.89 vs 9.08 pM; Fig. 2A, Table 2). For these studies, human MDA-MB-231 TNBC cells were also examined. In these cells, IC50S for ZG-126 and AM-193 were similar (0.92 vs 0.5 pM; Fig. 2B, Table 2), whereas those of DK-406, ZG-102 and ZG-132 were 2.43, 2.18 and 0.95 pM, respectively. Note that in these assays, 1 ,25D and SAHA alone or in combination did not display significant cytotoxicity at saturating 100 nM concentrations (Figs. 2A and 2B).Table 2. IC50S extracted from the in vitro cell viability assay

[0162] These assays were performed by Reaction Biology (Malvern, PA, USA) using standard in-house protocols. Briefly, ZG-126 was tested in 10-dose IC50 mode, in singlet, with 3-fold serial dilution starting at 100 pM. HDACi reference compound Trichostatin A (TSA) was tested in a 10-dose IC50, with 3-fold serial dilution starting at 10 pM or 1 pM, depending on the HDAC tested. HDAC reference compound TMP269 was tested in a 10-dose IC50, with 3-fold serial dilution starting at 10 pM. HDAC reference compound Quisinostat was tested in a 10-dose IC50 with 3-fold serial dilution starting at 1 pM. For HDACs 1 , 2, 3 and 6, a fluorogenic peptide from p53 residues 379-382 (RHKK(Ac)AMC) was used as a substrate. For HDACs 4, 5, 7, 9 and 11 , a fluorogenic HDAC Class Ila substrate Trifluoroacetyl Lysine was used. For HDAC8, a fluorogenic peptide from p53 residues 379-382 (RHK(Ac)K(Ac)AMC) was used as a substrate, whereas Ac-Spermidine-AMC was the substrate for HDAC10 assays.

[0163] After 6 h treatments, 4T1 , 4T07 or B16-F10 cells were solubilized in Lysis Buffer (20 mM Tris, pH 8, 150 mM NaCI, 1% Triton™ X-100, 3.5 mM sodium dodecyl sulfate (SDS), 13 mMdeoxycholic acid and proteins were separated on a 4-15% Tris / Glycine / SDS gel (BIO RAD). Then the proteins were transferred to a nitrocellulose membrane followed by blocking in 5% skim milk in Tris-buffered saline solution (TBS, 1X). After blocking, the membranes were incubated overnight at 4 °C with the primary antibodies against acetylated tubulin (Sigma Aldrich - #T7451), tubulin (Sigma Aldrich - #T9026), acetyl-histone H3(Lys9) (Merck Millipore - #07-352), acetyl-histone H3 (Lys27) (Abeam -#ab4729), total histone H3 (Cell Signaling Technology (CST) -#3638) and glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) (Abeam - #ab8245). Then the membranes were incubated with anti-mouse (CST - #7076) or anti-rabbit (CST - #7074) immunoglobulin G (IgG) horseradish peroxidase (HRP) linked secondary antibodies at recommended concentrations. Finally, signals were detected using clarity enhanced chemiluminescence (ECL) substrates (Bio-Rad) and ChemiDoc™ Imaging System. Changes in protein levels were quantified relative to control using Image Lab software (Version 6.0.1) after normalization to GAPDH. Western-blot experiments were carried out three times.

[0164] The potential for compounds as HDAC inhibitors can be rapidly tested in cells in culture by monitoring hyperacetylation of histone H3 and tubulin. H3 acetylation is controlled by multiple classI enzymes and class lib enzyme HDAC6 regulates tubulin acetylation. In 4T1 cells, AM-193 (10 pM) was a highly efficacious inducer of tubulin acetylation. Of the ZG compounds, ZG-126 was by far the most efficacious (Fig. 3A) at 10 pM and several-fold more efficacious than a saturating concentration of SAHA (100 nM) (Fig. 3A). ZG-126 also performed well at inducing histone H3 hyperacetylation at both lysine 9 and 27 (H3K9, H3K27; Figs. 3B-3C) in 4T1 cells, and was comparable or superior to AM-193. Similarly, in 4TO7 cells, ZG-126 induced higher levels of tubulin and histone hyperacetylation than SAHA and was as or more efficacious as ZG-102 and ZG-132 (Figs. 3D-3F).

[0165] As ZG-126 had the most favorable bifunctional profile of the ZG compounds, HDAC inhibition was further assessed in biochemical assays. IC50S were determined for all 11 class I, classII or class IV HDACs; i.e. those (unlike Sirtuins, class III) that are zinc metalloenzymes. Trichotomic A (TSA) was used as a potent and non-selective inhibitor of class 1 enzymes (HDACs 1-3 and 8), class lib enzyme HDACs 6 and class IV enzyme HDAC 11. The selective class II inhibitor TMP269 was used for class Ila enzymes HDACs 4, 5, 7 and 9, and quisinostat was used for the HDAC10 assay because it is a potent inhibitor of the enzyme. IC50S for ZG-126 ranged by 100-fold across different isoforms, from 0.63 pM for HDAC6 to 68 pM for HDAC4 (Table 3). The outlier was HDAC9, where no or very limited inhibition was observed to concentrations as high as 100 pM. Raw data for HDACs 1-3 and HDAC6, were chosen because they are key regulators of histone H3 acetylation and tubulin acetylation, respectively, are shown in Figs. 4A-4D, whereas data for other HDACs are in presented in Figs. 5A-5G. The sub-pM IC5o for HDAC6 and low pM IC50S for HDACs 1-3 are consistentwith the capacity of ZG-126 to induce tubulin and histone H3 hyperacetylation in living cells. The IC50 of 4.6 pM of ZG-126 for inhibition HDAC2 represents a modest improvement on that of AM-193 (7.2 pM), whereas the IC50 for HDAC6 inhibition was slightly higher (0.63 vs 0.3 pM). Taken together these data suggest that, with the exception of HDAC9, ZG-126 acts as a pan-HDAC inhibitor of varying potency.Table 3. IC50S for inhibition of purified HDACs 1-11 by ZG-126ND = not detected

[0166] ZG-126 was further tested in vitro, along with previously developed compounds in mouseB16-F10 melanoma cells, which are derived from tumors in C57BL / 6 mice. B16-F10 cells are responsive to 1 ,25D analogs45 and HDACi, and are widely used in preclinical studies of melanoma solid tumor growth and metastases. Although curable when treated early, melanoma is fatal when invasive. The American Cancer Society projected 97,610 new invasive melanoma cases in the US in 2023 and 7,990 deaths. Current treatment options include chemotherapy, surgery, immunotherapy or single agents targeting mutated genes such as B-RAF or C-KIT.49 The bifunctionality of ZG-126 was first characterized in B16-F10 cells. In contrast to 4T1 and 4TO7 cells, 1 ,25D and SAHA combined super-induced Cyp24a1 expression in the B16-F10 model, an effect that was mimicked by ZG-126 treatment (Fig. 6A). Unlike in TNBC cells (Figs. 3A-3F), DK-406 induced very high levels of tubulin acetylation and histone hyperacetylation. Hyperacetylation observed in the presence of ZG- 126 was comparable or greater than that induced by a saturating concentration of SAHA (100 nM) (Figs. 6B-6E), and its cytotoxicity was similar to that of AM-193 (Fig. 6F).

[0167] All animal experiments were carried out according to the McGill University Animal Care guidelines at the Lady Davis Institute. Briefly, 4T 1 cells (1 x 106cells per mouse) or B16-F10 cell (2.5 x 105per mouse) were implanted into the mammary fat pad of female Balb / c mice or subcutaneously in female C57BL / 6 mice, respectively. As soon as the tumors became palpable (day 7-14), treatments began by intraperitoneal (IP) injection of DMSO (vehicle), 1 ,25D and / or SAHA or the compounds every second day (250 pL total volume). Throughout the course of treatment (18 days), the primary tumor size was measured 5 times using a caliper, and tumor volume was calculated as (length x width2) / 2. Finally, mice were sacrificed, and lungs of 4T1 bearing mice were removed to evaluate the efficiency of treatments on lung metastases. As such, lungs were fixed in 10% Bouin’s fixative and surface lung metastases were counted using a stereomicroscope (Optimax; Leica). For MTD studies, Balb / c mice were given increasing daily doses of indicated compounds IP (0.5 mg / kg, 1.0 mg / kg, 1.5 mg / kg, 2.5 mg / kg, 5 mg / kg, 10 mg / kg). Once mice demonstrated any sign of discomfort (poor oral intake, lethargy, weight loss, inability to close mouth, drooling, pawing at mouth), the study was terminated.

[0168] The maximum tolerated dose (MTD) of compounds was tested by daily administration to Balb / c mice. Under this dosing regimen, the MTD of DK-406 was limited to 1.0 mg / kg (Table 4), whereas AM-193 proved to be less well tolerated, with an MTD of 0.5 mg / kg. In contrast, the MTDs of all three ZG compounds were tolerable at least 10 mg / kg.Table 4. Maximal tolerated daily doses of compounds as indicated

[0169] The efficacy of ZG compounds was then compared to other compounds in tumor models in vivo. The initial focus was on the B16-F10 melanoma which are syngeneic with C57BL / 6 mice. Efficacy of ZG-126 in vivo in this model was tested in two experiments. First, as IC50S for several HDACs were in the sub to low pM range, whereas optimal induction of Cyp24a1 (Vdr agonism) invitro was observed at 10 pM, it was important to determine if a potentially therapeutic dose of ZG- 126 would induce Vdr agonism in vivo. Thus, B16-F10 cells were implanted subcutaneously and, when tumors became palpable, were treated with two doses of ZG-126 (5 mg / kg) 24 h apart prior to harvesting RNA. Remarkably, in duplicate tumors from two animals, this led to robust induction of Cyp24a1 as well as the gene encoding the Vdr itself, which is autoregulated (Fig. 7A), confirming Vdr agonism of ZG-126 in vivo at a therapeutic dose. Fig 7Ai also illustrates that ZG126 also functions as an HDACi at well-tolerated therapeutic doses in vivo in B16-F10 tumors. In a second experiment, the antitumor efficacy of compounds was compared at ~0.5 MTD doses. ZG-126 (5 mg / kg daily) and AM- 193 (0.2 mg / kg daily) were tested along with 1 ,25D (0.25 pg / kg) and SAHA (80 mg / kg) alone or in combination, along with gemcitabine (50 mg / kg daily) as a positive control. All treatments were well tolerated, and body weights were unaffected (Fig. 7B). Compounds, 1 ,25D and SAHA together, and gemcitabine all produced a statistically significant reduction in tumor burden, with compounds being at least as efficacious as the gemcitabine positive control (Figs. 7C-7D).

[0170] The efficacy of AM- 193 at 0.2 mg / kg daily was then compared with that of ZG-126 at low and high doses (1.5 and 5 mg / kg, respectively) in the mouse 4T1 TNBC model, along with 1 ,25D (0.25 pg / kg) and SAHA (80 mg / kg) alone or in combination. As 4T 1 cells are aggressively metastatic, anti-metastatic activity was also assessed in the same experiment. All treatments were well tolerated, and, as in the B16-F10 model, none induced weight loss or other signs of stress over the course of the experiment (Fig. 8A). Treatments reduced primary tumor burden to varying degrees. The combination of 1 ,25D and SAHA did not appear to be more efficacious than either compound alone, producing a non-significant reduction in tumor volume of -20%. Notably, unlike in the B16-F10 model above, AM-193 (0.2 mg / kg) was less efficacious than high dose ZG-126 (5 mg / kg), which was the only treatment that produced a statistically significant reduction in tumor burden (-50%; Figs. 8B-8C).

[0171] Anti-metastatic activities were determined by counting surface lung metastases of duplicate samples using a stereomicroscope. Here again, high-dose ZG-126 proved to be the most efficacious, substantially reducing the size and reducing the surface metastases by almost four-fold (Fig. 8D). This degree of efficacy was remarkable, DK-406 reduced metastatic burden by —2-fold . The magnitude of the anti-metastatic effect of ZG-126 in these experiments is comparable to those observed with other therapeutic agents, such as cis-platin, and suggests that it is efficacious clinically in a neoadjuvant setting in reducing TNBC metastases. This result is significant, because, in the clinic, compounds like ZG-126 would likely be used after surgical removal of the primary tumor.

[0172] RNA extraction was performed with the FavorPrep™ tissue total RNA mini kit (FAVORGEN™ Biotech Corporation — FATRK 001) as per manufacturer’s instructions. cDNA wasobtained from 1 g of RNA using 5X All-in-One RT Mastermix (Applied Biological Materials (abm) Inc. — G485) and diluted 5 times. Quantitative polymerase chain reaction (qPCR) was performed with BrightGreen 2X q-PCR MasterMix (abm — MasterMix-LR-XL) on a Roche LightCycler™ 96 system. Expression of targeted genes were normalized to 18s. All primers are listed in Table 5.Table 5. Primer sequences for RT-qPCR

[0173] RNA-seq analysis performed in 4T1 cells previously performed revealed that 1 ,25D and compounds induced numerous changes in gene expression profiles. One of the signaling pathways enriched in treated cells was implicated in regulation of myeloid cell infiltration into the tumor microenvironment (TME), which is particularly relevant to TNBC. Expression of genes controlling macrophage migration was downregulated in compound-treated cells compared to their untreated counterparts. Here, the effect of ZG-126 on regulation of the expression of genes encoding cytokines and chemokines were analyzed regarding their involved in recruitment and polarization of macrophages. These include Cc / 2, Cc / 5, Ccl20 and CxcHO, all of which are implicated in promoting breast tumor growth or metastasis, and promoting the polarization of tumor-associated macrophages to an M2 anti-inflammatory phenotype. Treatment with either 1 ,25D or the present compounds inhibited the expression of Cc / 2, Cc / 5, Ccl20 and CxcHO (Figs. 9A-9E). In contrast, 1,25D and the present compounds induced the expression of the gene encoding interleukin-1 a (Figs. 9A-9E), a cytokine that polarizes macrophages to an inflammatory M1 phenotype.

[0174] Primary tumor tissues were fixed using 4% paraformaldehyde and subsequently dehydrated in sucrose solutions ranging from 10% to 30% for approximately 24 h each. The tissues were then embedded in base molds (Fisher Brand - #22363553) containing optimal cutting temperature (OCT) compound (Fisher Healthcare - #4585). The tissue blocks were placed immediately on dry ice until OCT solidified and then they were stored at -80 °C. Prior to immunofluorescence (IF) staining, sections of 5 to 10 pm were obtained using a Leica CM3050 S cryostat and SuperFrost™ Plus slides (Fisher Brand - #1255015). The tumor sections were then incubated with blocking buffer (phosphate buffered saline (PBS) containing 2% bovine serum albumin (BSA), 0.3% Triton X-100, 1 % FBS, and 10% goat serum) for 1 h at RT. To detect macrophages in the TME, the sections were incubated with a pan-macrophage rat monoclonal antibody, F4 / 80 (Abeam - #6640) diluted in blocking buffer overnight at 4°C. 16 h later, tissues were washed 3 times with 100 mM Tris-HCL and then incubated with a secondary goat anti-rat antibody (Invitrogen - #A11081) for 1 h at RT. The washing step was repeated followed by incubation with an M2 macrophage marker, Alexa-647 Arg1 (CST -#43279) for 90 min at RT. Subsequently, 4',6-diamidino- 2-phenylindole (DAPI; Invitrogen - #D3571) was used to counterstain the nuclei for 5 min at RT. Finally, the slides were mounted using Fluoromount-G™ medium (Invitrogen - #00495802) and coverslips (Fisher Scientific - #12545E) and imaged with a Zeiss LSM 710 confocal microscope withx20 objectives. Quantification was carried out by QuPath™ software (version 0.3.2). F4 / 80+ macrophages and F4 / 80+ Arg1+ M2 macrophages were classified based on their fluorescence signal.

[0175] To further probe the effects of 1 ,25D and SAHA, alone or in combination, or ZG-126 on macrophage infiltration into 4T1 tumors, IF imaging of tumor samples was performed. Treatment with 1 ,25D or ZG-126, but not SAHA, significantly reduced total macrophage infiltration into 4T1 tumors, as measured by F4 / 80 staining (Fig. 9E), consistent with effects of these compounds on gene expression. Moreover, ZG-126 treatment was the most efficacious at reducing the percentage of antiinflammatory M2 macrophages, as measured by Arginase 1 (Arg1) staining (Figs. 9F-9I, and Figs. 10A-10Q). Tumor-associated macrophages are among the first to infiltrate and the most frequent immune cells in the TME 63-65. Cancer cells tend to secrete chemokines and cytokines that favor differentiation of anti-inflammatory M2-polarized macrophages in the TME, which are immunosuppressive. Thus, the results strongly suggest that ZG-126 induces the formation of a less immunosuppressive microenvironment in 4T1 tumors.

[0176] This targeted effect indicates that ZG-126 would be efficacious when combined with immunotherapeutics. For example, co-targeting TAMs with ZG-126 and phagocytosis checkpoint blockades may reshape the TME and enhance reprogramming of TAMs toward a more tumoricidal phenotype. Notably, TAM-targeted strategies restore the anti-tumor effects of CD8+ T lymphocytes in TME by two means, first by suppressing secretion of chemokines (CCL2, CCL18, and CCL22), cytokines (IL-4, IL-10, and TGFP) and enzymes (COX2, MMPs, and ARG1) that negatively regulate T-cell cytotoxicity, and second by suppressing signaling by TAM-expressed ligands such as CD80 / CD86 and PD-L1 , which attenuate immune responses. Inhibition of PD-L1 and CD80 / CD86 is directly achieved through immune checkpoint blockade of their receptors using anti-CTLA-4 and anti- PD-1 antibodies. Combination of ZG-126 with immune checkpoint inhibitors could be an option to boost the immune function against tumor growth and metastasis.

[0177] In the present example, all statistics were calculated using GraphPad™ Prism (version 8). A parametric unpaired t-test was used for comparison of two groups in all in vitro experiments while a two-way ANOVA was used to examine the differences in primary tumor volume in the in vivo experiment. Statistical significance is indicated as follows: *p < 0.05, **p < 0.01 , ***p < 0.001 , and ****p < 0.0001.

[0178] In summary, the ZG series of bifunctional VDR agonist / HDACi hybrid molecules was synthesized and tested in vitro and in vivo. Replacing the non-polar ether linker in the hydroxamic sidechain of the AM series of compounds 33 with an amide substantially improved solubility without sacrificing bifunctionality. Indeed, ZG-126 was a full or super-agonist of the VDR and generallyinduced robust tubulin or histone hyperacetylation in multiple cell lines. ZG compounds were substantially better tolerated in vivo than AM- 193 or DK-406, and evidence was provided that ZG- 126 can induce robust VDR target gene expression in vivo. A high dose regimen of ZG-126 displayed anti-tumor activity in mouse melanoma and TNBC models along with impressive anti-metastatic activity in the mouse 4T1 TNBC model. Finally, ZG-126 treatment inhibited macrophage infiltration into 4T1 tumors and diminished by two-fold the proportion of M2-polarized immunosuppressive macrophages.EXAMPLE 2

[0179] The present compounds are designed based on the fusion of critical elements of the non- secosteroidal analog of 1 ,25D, LG190178, an easily synthesized VDR agonist, and the sidechains containing critical zinc-chelating hydroxamic acids of HDAC inhibitors (HDACi) such as SAHA and TSA. Original compounds retained the diarylpentane core of LG190178, which rendered the molecules quite hydrophobic. The AM series was created to introduce a more hydrophilic heterocycle into the core. Both the ZG series and the AC series were subsequently designed (see structures below) in an attempt to further improve compound solubility relative to AM-193 without substantially sacrificing bifunctionality. As with the AM series, the three compounds in each of the ZG (Example 1) and AC (described in this Example) series vary in lengths of the hydroxamic acid-bearing sidechains. The ZG series differs from the compound AM-193 by replacing the non-polar ether linker in the AM sidechain with a polar amide group. This improved solubility substantially as demonstrated in Example 1. The properties of the ZG compounds, including studies of in vivo efficacy in breast cancer and melanoma models are described in Example 1. The present Example focuses largely on the properties of compound AC-340, along with its comparison to ZG-126 and AM-193. In the AC compounds series, the pyridine heterocycle is replaced by a thiazole, i.e. a 6-membered ring is replaced with a more polar 5-membered ring (see structures below).

[0180] The basis of the AC series design was to further increase solubility by introducing the relatively hydrophilic thiazole heterocycle. This strategy is not straightforward, because, in other studies, replacement of the pyridine ring of the AM series with a pyrimidine produced compounds that were only partial VDR agonists at 10 pM (see Example 3).

[0181] One potential benefit of the AC structure would be improvement in the maximum tolerated dose (MTD) in vivo. That of AM-193 was poor (0.5 mg / kg), whereas that of ZG-126 was substantially improved (>7.5 mg / kg; see Example 1). However, the properties of AC compounds in this regard would be difficult to predict. ZG compounds are similar in structure to AM-193 except for the amide sidechain linker replacing the ether. AC compounds have a different heterocycle but the AM series ether in the sidechain.

[0182] The AC 339-341 series of compounds were prepared by alkylation of p- benzyloxyphenylacetonitrile using lithium diisopropylamide and ethyl iodide (Scheme 2). The resulting nitrile was transformed to a thioamide by treatment with potassium hydroxide followed by treatment with Lawesson’s reagent. The thioamide was condensed with 3-bromo-6-((terf- butyldimethylsilyl)oxy)-7,7-dimethyloctan-2-one to form the thiazole. After removal of the benzyl group by hydrogenolysis over palladium on carbon, a series of side chains were incorporated by alkylation with the appropriate haloester in the presence of potassium carbonate. Finally, treatment with hydroxylamine in the presence of potassium hydroxide furnished the hybrids.Scheme 2.

[0183] AC compounds were analyzed using the similar assays and protocols as the ZG compounds (see Example 1). Mouse B16-F10 and human A375 and SK-MEL-28 melanoma cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; 319-005-CL; WISENT Inc.) supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS; 098150; WISENT Inc.). Cells were then treated with DMSO (vehicle), 1 ,25D, SAHA, DK-406, AM-193, ZG-126 and AC compounds (339, 340, 341) for 6 or 24 h.

[0184] To determine the cytotoxicity of hybrid molecules, the assay was performed 24 h after treatment of B16-F10, A375 or SK-MEL-28 cells according to the manufacturer’s instructions (MTGIo, Promega -#G9712). Luminescence was measured using a 1420 Luminescence Counter Victor Light (PerkinElmer), normalized to control, and plotted. All samples were run in triplicate.

[0185] RNA extraction was performed with the FavorPrep™ tissue total RNA mini kit (FAVORGEN™ Biotech Corporation — FATRK 001) as per manufacturer’s instructions. cDNA was obtained from 1 pg of RNA using 5X All-in-One RT Mastermix (Applied Biological Materials (abm) Inc. — G485) and diluted 5 times. qPCR was performed with BrightGreen™ 2X q-PCR MasterMix (abm — MasterMix-LR-XL) on a Roche LightCycler™ 96 system. Expression of targeted genes were normalized to 18s.

[0186] Purified HDAC2 and HDAC6 were purchased from Cayman Chemical. Boc-Lys(Ac)-7- amino-4-methylcoumarin (BocLys(Ac)-AMC) was used as substrate for the HDAC assays or a similar substrate could be used. Substrate solution was prepared as follows: Boc(Lys-Ac)-AMC was dissolved in DMSO and diluted with HDAC buffer (15 mM tris-HCI [pH 8.1], 250 pM EDTA, 250 mM NaCI, 10% glycerol) to give 1 mM solutions containing 1.7% DMSO. Trypsin was used to stop the reaction, releasing free AMC. The trypsin solution was prepared as follows: trypsin was dissolved in HDAC buffer to give a concentration of 10 mg / mL. Release of AMC was monitored by measuring the fluorescence at 460 nm (lex = 390 nm) with a microplate reader (SpectraMax™ Gemini from Molecular Devices) at 37°C. The AMC signals were recorded against a blank with buffer, substrate and trypsin but without the enzyme. All experiments were carried out at least in triplicate.

[0187] For HDAC inhibition assays, inhibitor diluted in 50 pL of HDAC buffer was mixed with 10 pL of diluted enzyme solution in HDAC buffer at room temperature. The HDAC reaction was started by adding 40 pL of substrate solution in HDAC buffer followed by 60 min of incubation with stirring at 37 C. The reaction was stopped by adding 100 pL of trypsin solution. After a 30 min incubation with stirring at 37 °C, the release of AMC was monitored by measuring the fluorescence.

[0188] Western blot analysis was performed after 6 h treatments, B16-F10, A375 and SK-M EL- 28 cells were solubilized in lysis buffer (20 mM Tris, pH 8, 150 mM NaCI, 1 % Triton X-100, 3.5 mM SDS, 13 mM deoxycholic acid and proteins were separated on a 4-15% Tris / Glycine / SDS gel (BIO RAD). Then the proteins were transferred to a nitrocellulose membrane followed by blocking in 5% skim milk in Tris-buffered saline solution (TBS, 1X). After blocking, the membranes were incubated overnight at 4 °C with the primary antibodies against acetylated tubulin (Sigma Aldrich - #T7451), tubulin (Sigma Aldrich -#T9026), acetyl-histone H3(Lys9) (Merck Millipore -#07-352), acetyl-histone H3 (Lys27) (Abeam - #ab4729), total histone H3 (Cell Signaling Technology (CST) - #3638) and GAPDH (Abeam - #ab8245). Then the membranes were incubated with anti-mouse (CST - #7076) or anti-rabbit (CST - #7074) IgG HRP-linked secondary antibodies at recommended concentrations. Finally, signals were detected using Clarity ECL substrates (Bio-Rad) and ChemiDoc Imaging System. Changes in protein levels were quantified relative to control using Image Lab software (Version 6.0.1) after normalization to GAPDH. Western-blot experiments were carried out three times.

[0189] B16-F10 cells were prepared in triplicate and RNA was extracted after 24 h treatment(vehicle, 1 ,25D, 100 nM; SAHA, 100 nM; 1.25D + SAHA, 100 nM each; AM-193, ZG-126 or AC-340, 10 pM each). After RNA extraction, all RNAs had a concentration higher than 100 ng / pL and a ratio 260 / 280 around 2 and were sent to the McGill Genome Centre. McGill Genome Centre performed a quality control before sequencing in paired-end at 50 M reads by Illumina NovaSeq™ 6000 S4 PE100. After sequencing, the FASTQ files were used for the analysis performed on Galaxy™. First, the trimmomatic tool was used to remove the adapters from the reads. Then, using the alignment tool, HISAT2, the reads were mapped to the mouse genome. In the next step, the counted reads were annotated and finally the list of differentially expressed genes (DEGs) for each treatment was obtained using the pre-installed R package called Limma™.

[0190] VDR agonism was tested by induction of the Cyp24a1 gene. Cyp24a1 encodes the enzyme that initiates catabolic breakdown of 1 ,25D, and its gene expression is exquisitely sensitive to the presence of agonist-bound VDR. Unexpectedly, AC340 displayed VDR superagonist activity on the Cyp24a1 gene at 10'5M in mouse B16-F10 melanoma cells, while other compounds exhibited including AC-339 and AC-341 (Fig. 11A). In multiple experiments, Cyp24a1 induction was >10-fold higher than that observed with 100 nM 1 ,25D, a saturating concentration and several-fold higher than other compounds. The VDR superagonist activity of AC-340 was entirely unexpected, and at the moment is unexplained.

[0191] The IC50 of AC-340 for HDAC2 inhibition in vitro was slightly higher than those of AM-193 and ZG-126, but its IC50 for HDAC6 inhibition in vitro was —10-fold lower (Table 6). Efficacy ofinduction of protein hyperacetylation at 10'5M in B16-F10 cells by AC-340 was comparable to or superior to other compounds (Figs. 11 B-11 E). Of the AC compounds, AC-340 thus appeared to be the most robustly bifunctional in B16-F10 cells and was selected to be tested further. Similar results for tests of bifunctionality of AC-340 were obtained in two human melanoma cell lines A375 and SKIM EL-28 (Figs. 12A-12E and 13A-13E). VDR superagonism of AC-340 was observed in both cell lines at 10-5M and by ZG-126 in SK-M EL-28 cells (note that AC-340 induced extensive cell death at the highest concentration tested in SK-M EL-28 cells (3x10'5M)). AC-340 (and ZG-126) was also highly efficacious in protein hyperacetylation assays in human melanoma cell lines and its efficacy was generally superior to that of AM-193.Table 6. IC50S for inhibition of purified HDAC2 or HDAC6 in vitro

[0192] B16-F10 cells were treated with DMSO, 1.25D, SAHA (100 nM each) or the combination of both, AM-193, ZG-126 or AC-340 (10 pM each). After 24 h, cells were trypsinized and collected in Eppendorf tubes followed by spinning down and aspirating the medium. Cells were then resuspended in ice cold PBS, counted and stained for viability detection (eBioscience -#65-0865-14). After 20 min incubation at 4°C in the dark, the cells were centrifuged at 1500 rpm for 5 minutes at 4°C, then the supernatant was flicked and the cells were resuspended in 50 pL of surface stain cocktail containing P2M and H2KbDb antibodies diluted in cold FACS buffer (PBS +1mM EDTA +2% FCS) and incubated for 30 min at 4°C in the dark. Finally, the cells were washed at 2000 rpm for 5 minutes at 4°C and resuspended in 250 pL FACS buffer and transferred into FACS tubes. Samples were acquired using a BD LSR Fortessa and analyzed with FlowJo along with the single stain controls for each channel used by adding 0.5 pL of antibody to 1 drop of beads.

[0193] AC compounds (and ZG-126) were cytotoxic in the MTGIo assays in the three melanoma cell lines, with IC50S comparable to that of AM-193 (Figs. 14A-14C). The only exception was ZG-126 in SK-MEL-28 cells, which appeared less cytotoxic. The bifunctionality and potential for therapeutic efficacy of compounds were further probed in an RNAseq study in B16-F10 cells as outlined in Fig. 14A. Cells were treated for 24h under each condition (1,25D, 100 nM; SAHA, 100nM; compounds 10’5M). This timeframe was not chosen to identify primary target genes, but to reveal cellular pathways that would be affected by longer-term exposure to compounds. It is clear from the results that B16- F10 cells are substantially more responsive to 1.25D+SAHA than to each compound in isolation and even more responsive to compounds. Remarkably, AC-340 treatment produced 3 times more differentially expressed genes (DEGs) than AM-193, with ZG-126 producing an intermediate number (Table 7). The profiles of DEGs identified in AM-193- or ZG-126-treated cells largely overlapped with DEGs in the presence of AC-340 (Figs. 15A-15B). The varying effects of each treatment on the numbers of regulated genes and the fold changes observed are reflected in plots of log-fold changes and volcano plots (Figs. 16A-16L).Table 7. Number of differentially expressed genes (DEGs) observed under each treatment condition

[0194] The enhanced response of B16-F10 cells to AC-340 may at least partly reflect the VDR superagonist activity observed in Cyp24a1 I induction assays (Figs. 11A-11 E). To examine this further, the fold regulations of the top 50 most induced genes by 1 ,25D were aligned with those observed with AC-340 treatment. In 49 of 50 cases, AC-340 superinduced their expression, often several folds (Fig. 17A). Relative regulation of three of these was validated by real time (RT) qPCR (Figs. 17B-17D). In all three cases, AC-340 treatment induced their expression several-fold more than 1 ,25D. For two of the three genes, the fold regulation by AC-340 was also substantially higherthan those observed with AM-193 and ZG-126, which would be consistent with the greater numbers of DEGs identified in AC-340-treated cells. The present compounds in general and AC-340 in particular were also more efficacious inhibitors of gene expression. One notable example is the gene encoding the melanoma oncogene cKit, which is repressed ~2-fold by 1.25D+SAHA and 10-fold or more by the present compounds (Figs. 17E-17I).

[0195] Lists of DEGs with a p value of < 0.05 and a two-fold cut-off for each comparison were analyzed with Ingenuity Pathway Analysis or IPA (QIAGEN Inc.) to identify enriched diseases and biological functions. IPA uses Fisher’s exact test to determine a probability value to display the association between each gene in the list and IPA-curated pathways and biological functions. A log p-value cut-off of 1.3 was considered statistically significant overrepresentation of genes in a disease or biological function. Furthermore, IPA employs a z-score of greater than 2 and less than - 2 to indicate significant predicted activation and inhibition states of functions, respectively.

[0196] A pathways analysis was performed to determine the major biochemical pathways and cellular functions regulated by AC-340 in B16-F10 cells. The most strongly affected pathways were all downregulated, and the vast majority of those were related to cell cycle regulation, and regulation of DNA replication and mitosis, consistent with cell cycle arrest induced by AC-340 treatment (Table 8). Numerous gene networks were also downregulated (Table 9), and many were consistent with the pathways analysis, as they include cMYC, its cofactor Max and several members of the E2f family of transcriptional activators, all implicated in G1-S cell cycle transition. In contrast, a pathway controlled by E2f6, which, unlike other E2F members, is a transcriptional repressor, and was upregulated. Regulation by AC-340 and the present compounds of several genes implicated in cell cycle regulation was validated in Figs. 18A-18G. The only upregulated gene tested encoded Mxd1 , which heterodimerizes with cMYC cofactor Max and acts as a cMYC antagonist. This is consistent with a profound inhibition of cMYC function. The present compounds in general and AC-340 in particular were more efficacious than 1 ,25D and / or SAHA. The regulation of a group of gene representative of melanocyte differentiation was also tested, all of which were selectively downregulated by the present compounds (Figs. 19A-19D).Table 8. Major biochemical pathways regulated by AC-340 in B16-F10 cellsTable 9. Gene networks downregulated by AC-340 treatment of B16-F10 cells

[0197] The bioinformatics analysis provided evidence that AC-340 upregulated pathways consistent with signaling by 1 ,25D (calcitriol) and SAHA (vorinostat), demonstrating its bifunctionality (Table 10). The most strongly regulated pathway was that controlled by p53 (TP53) (Table 11). Importantly, several criteria indicate that AC-340 also generated an expression profile consistent with rendering immunologically cold B16-F10 cells more susceptible to immune checkpoint inhibitor (ICI) therapy. AC-340 regulated genes consistent with activated IFNy (IFNG) and Nf-kb signaling (NF-kB activators IKKp and IKKy), both of which are hallmarks of melanoma responses to ICI. In addition, AC-340 upregulated a pathway consistent with activated transglutaminase 2 (TGM2) signaling, another marker of antitumor immunity in melanoma, and expression of the Tgm2 gene was superinduced by AC-340.Table 10. AC-340 induces both calcitriol and SAHA (vorinostat) signaling pathwaysTable 11. Gene networks upregulated by AC-340 treatment of BF16-F10 cells

[0198] AC-340 induced expression of multiple genes implicated in antigen presentation. These included MHC class I genes H2-d1 and H2-k1, among others, as well as that encoding MHC class I cofactor p2-microglobulin (Figs. 20A-20F). AC-340 was a generally more efficacious an inducer of these genes than other compounds (Figs. 20G-20H). AC-340 and ZG-126 repressed genes encoding checkpoint inhibitors Pd-11 and Pvr (Cd155), two proteins that combine to determine sensitivity to Pd- L1 blockade in melanoma. The upregulation by the present compounds of MHC class I components (Figs. 21A-21 B) and p2-microglobulin proteins (Figs. 21C-21 D) on B16-F10 cells was confirmed by flow cytometry. The present compounds generally performed similarly, with the exception of ZG-126, which was a less efficacious inducer of p2-microglobulin than AM-193 or AC-340.

[0199] All animal experiments were carried out according to McGill University Animal Care guidelines by at the Lady Davis Institute. For MTD studies, Balb / c mice were given increasing daily doses of indicated compounds IP (0.5, 1.0, 1.5, 2.5, 5, 10 or 15 mg / kg). Once mice demonstrate any sign of discomfort (poor oral intake, lethargy, weight loss, inability to close mouth, drooling, pawing at mouth), the study was terminated.

[0200] Finally, the maximum tolerated doses (MTDs) of AC-340 was compared to those of AM- 193 and the ZG series of compounds in Balb / c mice (Table 12). AC-340 was tolerated to at least 15 mg / kg, which was superior to the ZG compounds, and several fold higher than that of AM-193 and DK-406, a precursor of the ZG compounds containing the original diarylpentane core. Similar MTDs were obtained for AC-339 and AC-341.Table 12. Maximal tolerated daily doses of compounds

[0201] Both (sets of) ZG and AC compounds are much better tolerated in vivo than AM-193. The MTD for AM-193 in Balb / c mice under the dosing schedule used was 0.5 mg / kg, whereas those of the ZG series were 10 mg / kg, a 20-fold improvement. This led to superior efficacy of high dose lead ZG compound ZG-126 as both an anti-tumor and antimetastatic agent in the mouse 4T1 triplenegative breast cancer model. AC compounds are therefore better tolerated than AM-193 (>15 mg / kg).

[0202] The markedly reduced toxicity of the ZG and AC compounds could not have been predicted from their structural modifications or their improved solubility. Prior to their synthesis, it was known that the solubility of AM-193 was improved over DK-406, which did not have a heterocyclic core, but DK-406 was somehow better tolerated in vivo. In addition, ZG compounds differ from AM- 193 in the HDACi sidechain (amide vs ether), whereas AC-340 differs in the heterocycle. Accordingly, the results are unexpected and surprising.

[0203] Indeed, it was completely unexpected that AC-340 performed as a VDR superagonist in human and mouse melanoma cell lines. This was initially observed by examining induction of theCyp24a1 gene, but was confirmed in RNAseq studies in B16 / F10 melanoma cells. B16 cells respond transcriptionally to 1.25D+SAHA better than either compound individually. The present compounds induce even more dynamic changes, with AC-340 producing the biggest transcriptional response followed by ZG-126 and AM-193. The magnitude of these differences could not have been predicted. This is consistent for both transcriptional activation and repression of target genes, with AC-340 being an even stronger repressor of some genes than AM or ZG.

[0204] AC-340 appears to generally induce greater tubulin and histone hyperacetylation in melanoma cell lines relative to AM-193. This was also not predictable based purely on structural differences.

[0205] Based on the structural modifications incorporated into the ZG and AC compounds, it was impossible to predict the magnitude of the transcriptional responses of B16-F10 cells to ZG-126 (>2,000 DEGs) and AC-340 (>3,000 DEGs) relative to AM-193 (>1 ,000 DEGs), and 1,25D and / or SAHA. It was also impossible to predict the extent to which the compounds would regulate different biochemical pathways and cellular processes in B16-F10 cells. Most noteworthy among these are the effects of AC-340 (and to a lesser extent ZG-126) on antigen presentation and the regulation of genes consistent with rendering immunologically cold B16-F10 cells more responsive to immune checkpoint inhibitor therapy. Based on these findings, the AC and LG compounds are an improvement as compounds for combination therapy with immune checkpoint inhibition.

[0206] In the present example, statistics were calculated using GraphPad Prism (version 8). A parametric unpaired t-test was used for comparison of two groups in all in vitro experiments while a two-way ANOVA was used to examine the differences in primary tumor volume in the in vivo experiment. Statistical significance is indicated as follows: *p < 0.05, **p < 0.01 , ***p < 0.001 , and ****p < 0.0001.EXAMPLE 3

[0207] The AC series of compounds was expanded by incorporating a pyrimidine ring to produce compounds AC-455, AC-454 and AC-451 following the protocol of Example 1 while modifying the synthesis of the biaryl section (Scheme 3). Briefly, 2-(4-bromophenyl)acetonitrile was alkylated by treatment with lithium diisopropylamide and ethyl iodide. Subsequent trimethylaluminum promoted addition of ammonia provided an amidine which could be condensed with ethyl 2-acetyl-5- (benzyloxy)-6,6-dimethylheptanoate in the presence of sodium methoxide to provide a pyrimidone. Carboxylation of the aryl bromide with CO, Pd(OAc)2 and methanol, followed by conversion of the pyrimidone to a pyramidine chloride with POCI3 and reduction with Pd(OH)2 and H2 afforded themethyl ester of the core of the pyrimidine substrates. These could then be converted as described for ZG hybrids (see above).Scheme 3.

[0208] VDR agonism was determined by evaluating the induction of the Cyp24a1 gene. Agonism was assessed initially in mouse 4T1 cells. AC compounds induced Cyp24a1 expression (Fig. 22). As can be seen in the figure, the activity was not deemed sufficient and no further experimentation was done on these three compounds.(82) (114)Ethyl pivaloylacetate (114)

[0209] To a solution of pinacolone (0.5 mL, 4 mmol, 1 equiv.) in dry THF (20mL, 0.2M) was added NaH (800mg, 20 mmol, 60% dispersion in mineral oil, 5 equiv.) and then diethyl carbonate (0.98 mL, 8 mmol, 2 equiv.). Refluxed the mixture overnight. Cooled the reaction to 0°C and quenched with ice water (15 mL). Extracted with ethyl acetate (15 mL, 3x). organic layers combined and dried over anhydrous sodium sulfate. Concentrated under reduced pressure and purified via FCC (5% ethyl acetate in hexanes) to give 114 as a colourless clear oil (680 mg, 98% yield). Physical and spectral data were in accordance with literature data.114 1154,4-Dimethylpentane-1 ,3-diol (115)

[0210] To a suspension of lithium aluminum hydride (146mg, 3.85mmol, 2.5 equiv.) in dry THF (10.3 mL, 0.15M), was added 118 (265 mg, 1.54mmol, 1 equiv.) in dry THF (5 mL) over 30 minutes at 0°C. Let the reaction warm to room temperature overnight. Then cooled to 0°C and slowly added

[0211] 0.14mL water, then added 0.14mL of 1M NaOH (aq.), and then added 0.44mL water.Warmed to room temperature and stirred for 15 minutes. Added anhydrous magnesium sulfate. Stirred for 15 minutes and filtered through celite. Concentrated under reduced pressure and purified by FCC (50% diethyl ether in hexanes to 100% diethyl ether). Gave 115 as a white solid (116 mg, 57% yield). Physical and spectral data were in accordance with literature data.148 1H NMR (500 MHz, CDCI3) 6 3.97 - 3.80 (m, 2H), 3.51 (ddd, J = 10.7, 3.9, 2.1 Hz, 1 H), 2.39 (d, J = 5.4 Hz, 1 H), 2.23 (d,J= 3.9 Hz, 1 H), 1.80 - 1.69 (m, 1 H), 1.69 - 1.58 (m, 1 H), 0.92 (s, 9H). HRMS (ESI+) calculated for C7H16O2 [M+Na]+: 155.1043, found 155.1046.115 1164-(Tert-butyl)-2-phenyl-1 ,3-dioxane (116)

[0212] To a solution of 119 (100 mg, 0.75 mmol, 1 equiv.) in dichloromethane (3.8 mL, 0.2M) was added benzaldehyde dimethyl acetal (136 pL, 0.91 mmol, 1.2 equiv.) and (+)-CSA (18 mg, 0.075 mmol, 0.1 equiv.) at room temperature. Let react overnight. Quenched with saturated aqueous sodium bicarbonate (5 mL). Extracted with dichloromethane (5 mL, 3x). Purified via FCC (5% diethyl ether in hexanes) to give 116 as a pale-yellow oil (145 mg, 94% yield). IR (thin film) v 2958, 2847, 1725, 1452 cm-1.1H NMR (500 MHz, CDCI3) 67.59 - 7.47 (m, 2H), 7.43 - 7.32 (m, 3H), 5.53 (s, 1 H), 4.32 (ddd, J = 11 .3, 5.0, 1.5 Hz, 1 H), 4.05 - 3.89 (m, 1 H), 3.48 (dd, J = 11 .5, 2.3 Hz, 1 H), 1.89 (dddd, J = 13.1 , 12.2, 11.4, 5.0 Hz, 1 H), 1.49 (dtd, J = 13.1 , 2.5, 1.5 Hz, 1 H), 1.00 (s, 9H).13C NMR (126 MHz, CDCI3) 6 139.28, 128.48, 128.10, 126.00, 101.01 , 84.78, 67.33, 34.12, 25.71 , 25.66. HRMS (ESI+) calculated for Cl4H20O2 [M+Na]+: 243.1356, found 243.1347.116 1173-(Benzyloxy)-4,4-dimethylpentan-1-ol (117)

[0213] To a solution of 116 (136 mg, 0.62 mmol, 1 equiv.) in dry dichloromethane (3.9 mL, 0.16 M) was added a solution of DIBAL-H (1.65 mL, 25 wt% in toluene) at -20°C. Stirred for 20 minutes then gradually warmed to room temperature. Stirred overnight then cooled to 0°C and slowly added 0.03 mL water, then added 0.03 mL of 1 M NaOH (aq.), and then added 0.07 mL water. Warmed to room temperature and stirred for 15 minutes. Added anhydrous magnesium sulfate. Stirred for 15minutes and filtered through celite. Extracted with ethyl acetate (5 mL, 3x). Dried over anhydrous sodium sulfate. Concentrated and purified by FCC (30% diethyl ether in hexanes) to give 117 as a pale-yellow oil (137 mg, quantitative yield). IR (thin film) v 3357, 2957, 2870 c r1.1H NMR (500 MHz, CDCI3) 67.41 - 7.34 (m, 4H), 7.33 - 7.29 (m, 1 H), 4.70 (d, J = 11.2 Hz, 1 H), 4.65 (d, J = 11 .2 Hz, 1 H), 3.90 - 3.72 (m, 2H), 3.30 (dd, J = 9.9, 3.0 Hz, 1 H), 1.85 (dddd, J = 14.4, 7.6, 5.5, 3.0 Hz, 1 H), 1.74 (dddd, J = 14.6, 9.9, 6.0, 5.0 Hz, 2H), 0.99 (s, 9H).13C NMR (126 MHz, CDCI3) 6 138.82, 128.40, 127.63, 127.58, 85.99, 75.17, 61.08, 36.11 , 33.58, 26.43. HRMS (ESI+) calculated for Cl4H22O2 [M+Na]+: 245.1512, found 245.1507.117 118(((1-Bromo-4,4-dimethylpentan-3-yl)oxy)methyl)benzene (118)

[0214] To a solution of 117 (137 mg, 0.62 mmol, 1 equiv.) and triphenylphosphine (170 mg, 0.65 mmol, 1.05 equiv.) in dry DCM (1 mL) at 0°C, added NBS (116 mg, 0.65 mmol) portion wise. Stirred at room temperature for 2h. Washed with water (5 mL, 3x), then brine (5 mL), dried organic layer over anhydrous Na2SO4. Concentrated and purified by FCC (2% diethyl ether in hexanes) to give 118 as a yellow oil (165 mg, 94% yield). IR (thin film) v 2957, 2869, 1496, 1454 cm’1.1H NMR (500 MHz, CDCI3) 6 7.41 - 7.35 (m, 4H), 7.33 - 7.29 (m, 1 H), 4.73 (d, J = 11.2 Hz, 1 H), 4.68 (d, J = 11.2 Hz, 1 H), 3.67 - 3.55 (m, 1 H), 3.55 - 3.47 (m, 1 H), 3.32 (dd, J = 8.1 , 4.1 Hz, 1 H), 2.10 - 2.00 (m, 2H), 0.99 (s, 9H).13C NMR (126 MHz, CDCI3) 6 138.89, 128.39, 127.54, 127.48, 85.61 , 75.58, 35.99, 34.70, 32.15, 26.45. HRMS (ESI+) calculated for Cl4H2lOBr [M+Na]+: 307.0668, found 307.0656.118 119Ethyl 2-acetyl-5-(benzyloxy)-6,6-dimethylheptanoate (119)

[0215] Added ethyl acetoacetate (94) (0.25 mL, 1.95 mmol, 2 equiv.), 118 (280 mg, 0.98 mmol, 1 equiv.), potassium carbonate (407 mg, 2.94 mmol, 3 equiv.), KI (162 mg, 0.98 mmol, 1 equiv.), DMF (1 mL) and acetone (13 mL) to a round bottom flask. Stirred at 70°C overnight. Cooled to roomtemperature and removed volatiles. Added water (15 mL) and extracted with ethyl acetate (10 mL). Dried organic layer over anhydrous Na2SO4, concentrated under reduced pressure and purified by column chromatography (10% ethyl acetate in hexanes) to give 119 as a clear colourless oil (206 mg, 63% yield). Inseparable mixture of isomers, the diastereomeric ratio (1 :1) was determined by NMR. IR (thin film) v 2957, 2869, 1737, 1713 cnv1.1H NMR (500 MHz, CDCI3) 5 7.40 - 7.31 (m, 4H), 7.29 (s, 1 H), 4.70 - 4.57 (m, 2H), 4.27 - 4.14 (m, 2H), 3.45 - 3.33 (m, 1 H), 3.02 (ddd, J = 8.8, 2.8, 1.5 Hz, 1 H), 2.20 (d, J = 12.0 Hz, 3H), 2.15 - 2.01 (m, 1 H), 1.93 - 1.81 (m, 1 H), 1.54 (74dd, J = 16.4, 8.2, 5.2, 2.5 Hz, 1 H), 1.50 - 1.35 (m, 1 H), 1.28 (td, J = 7.1 , 4.8 Hz, 3H), 0.98 - 0.90 (m, 9H).13C NMR (126 MHz, CDCI3) 6 203.19, 169.84, 169.77, 139.04, 139.01 , 128.32, 128.30, 127.54, 127.51 , 127.43, 127.39, 88.13, 87.88, 75.12, 74.93, 61.33, 61.32, 60.24, 59.99, 36.21 , 36.19, 29.09, 28.95, 28.90, 28.56, 26.43, 26.12, 26.06, 14.14, 14.12. HRMS (ESI+) calculated for C20H30O4 [M+Na]+: 357.2036, found 357.2031.2-(4-Bromophenyl)-2-ethylbutanenitrile (62)

[0216] To a solution of diisopropylamine (2.16 mL, 15 mmol, 2.9 equiv.) in anhydrous THF (27 mL) at -78°C was added n-butyl lithium (5.7 mL, 2.5 M in hexanes). Let stir for 1 minute. A solution of 63 (1 g, 5.1 mmol, 1 equiv.) in 9 mL of anhydrous THF was added dropwise via cannula. Stirred at -78°C for 15 minutes. Ethyl iodide (1.2 mL, 15 mmol, 2.9 equiv.) in 19 mL anhydrous THF was added dropwise to the reaction mixture via cannula. Stirred for 15 minutes at -78°C, then 1 hour at 0°C. Monitored reaction by TLC. Quenched with 1 M NH4CI (aq.) (30 mL) and extracted with ethyl acetate (30 mL). Washed organic layer with brine (30 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure. Purified via FCC (15:1 mixture of hexanes : ethyl acetate) to give 62 as a white solid (0.97 g, 75% yield). IR (thin film) v 2966, 2934, 2877, 2233, 1489 cm’1.1H NMR (500 MHz, CDCI3) 6 7.69 - 7.49 (m, 2H), 7.38 - 7.17 (m, 2H), 2.06 (dq, J = 14.7, 7.4 Hz, 2H), 1.97 - 1.82 (m, 2H), 0.93 (t, J = 7.4 Hz, 6H).13C NMR (126 MHz, CDCI3) 6 137.26, 131.97, 127.94, 121.83, 121.63, 49.58, 33.77, 9.66. HRMS (APCI+) calculated for Ci2Hi4NBr [M+H]+: 252.0382, found 252.0385.2-(4-Bromophenyl)-2-ethylbutanimidamide hydrochloride (60)

[0217] To a stirring solution of NH4CI (513 mg, 9.6 mmol, 2.5 equiv.) in dry toluene (4.8 mL), was added AIMes (4.8 mL, 2.0 M in toluene) dropwise at 0°C. let stir for 1 hour. Then 62 (969 mg, 3.84 mmol, 1 equiv.) was dissolved in 7.7 mL anhydrous toluene and added to the solution dropwise. Heated to 100°C for 5 days. Cooled to room temperature and slowly poured into a slurry of silica and DCM (75 mL). Stirred for 5 minutes. Filtered and washed with MeOH (75 mL). Combined filtrate and wash and concentrated. Added 2.3 mL of 3N HCI in EtOH and 51 mL anhydrous ether. Let sit in freezer overnight. Purified via FCC (5% MeOH in DCM) to give 63 as a gummy yellow solid (598 mg,51% yield). IR (thin film) v 3196, 2970, 2890, 1667, 1489 cm’1.1H NMR (500 MHz, DMSO) 5 9.15 (s, 2H), 8.65 (s, 2H), 7.90 - 7.56 (m, 2H), 7.33 - 7.04 (m, 2H), 2.00 (dp, J = 31.5, 7.1 Hz, 4H), 0.67 (t, J = 7.3 Hz, 6H).13C NMR (126 MHz, DMSO) 5 175.07, 140.82, 131.85, 129.81 , 121.21 , 51.82, 49.06, 25.71 , 8.04. HRMS (ESI+) calculated for Ci2Hi7N2Br [M+H]+: 269.0648, found 269.0645.(1 11 ) (119) (120)5-(3-(Benzyloxy)-4,4-dimethylpentyl)-2-(3-(4-bromophenyl)pentan-3-yl)-6-methylpyrimidin- 4(3H)-one (120)

[0218] Added sodium metal (30 mg, 1.3 mmol, 1.3 equiv.) to 1.8 mL methanol at 0°C. Then added 111 (300 mg, 0.98 mmol, 1.1 equiv.) at room temperature, then added 113 (298 mg, 0.89 mmol, 1 equiv.). Stirred at reflux overnight. Cooled to room temperature and removed volatiles. Added water (1 mL) and acidified to pH = 2-3 with 1M HCI (aq.). Then extracted with ethyl acetate (1 mL, 4x), dried over anhydrous Na2SO4 , and concentrated under reduced pressure. Purified by FCC (10% ethyl acetate in hexanes) to give 120 as a pale-yellow gummy solid (61% yield, 294 mg). IR (thin film) v 3142, 2965, 2875, 1636, 1592, 1452 cm’1.1H NMR (500 MHz, CDCI3) 6 10.06 (s, 1H), 7.49 - 7.42 (m, 2H), 7.42 - 7.33 (m, 4H), 7.30 (d, J = 7.3 Hz, 1 H), 7.10 - 7.00 (m, 2H), 4.82 - 4.65 (m, 2H), 3.13 (dd, J = 8.7, 2.8 Hz, 1 H), 2.65 (td, J = 12.4, 5.0 Hz, 1 H), 2.52 (td, J = 12.3, 4.8 Hz, 1H), 2.34 (s, 3H), 2.25 - 2.12 (m, 4H), 1.76 (ddp, J = 14.4, 7.7, 2.7 Hz, 1 H), 1.57 (dddd, J = 13.6, 11.6, 8.6, 4.9 Hz, 1 H), 0.99 (s, 9H), 0.66 (t, J = 7.4, 6H).13C NMR (126 MHz, CDCI3) 6 163.45, 160.14, 159.60, 142.17, 139.42, 131.50, 129.32, 128.28, 127.42, 127.31 , 122.77, 121.01 , 88.60, 74.84, 51.71 , 36.28, 29.77, 26.85, 26.53, 23.77, 21.76, 7.96. HRMS (ESI+) calculated for C30H39O2N2Br [M+Na]+: 539.2268, found 539.2270.Ethyl 5-((5-(3-(benzyloxy)-4,4-dimethylpentyl)-2-(3-(4-bromophenyl)pentan-3-yl)-6- methylpyrimidin- 4-yl)oxy) pentanoate (130)

[0219] Nil2 xH2O (1 mg, 0.0022 mmol, 0.05 equiv.), 4,4'-dimethoxy-2,2'-bipyridine (0.5 mg, 0.0021 mmol, 0.05 equiv.), sodium iodide (28.5 mg, 0.190 mmol, 0.25 equiv.), and 120 (23 mg, 0.042 mmol, 1 equiv.) were transferred to a 1-dram vial equipped with a magnetic stir bar. DMPU (1 ,3- dimethyl- 3,4,5,6- tetrahydro-2(1 H)-pyrimidinone, 0.17 mL), pyridine (1 pL, 0.0022 mmol, 0.05 equiv.), ethyl 5-bromovalerate (7 pL, 0.042 mmol, 1 equiv.), and zinc dust (5.5 mg, 0.085 mmol, 2 equiv.) were added. The reaction vial was capped, and the green solution was stirred at room temperature for approximately 5 min before heating to 60 °C in a reaction block. Upon completion, the reaction mixture was directly applied to the top of a chromatography column. Purified the reaction mixture by FCC (10% ethyl acetate in hexanes) to give 121 as a yellow solid (5.6 mg, 20% yield).1H NMR (500 MHz, CDCI3) 5 7.42 - 7.32 (m, 6H), 7.32 - 7.29 (m, 1 H), 7.17 - 7.12 (m, 2H), 4.73 - 4.60 (m, 2H), 4.25 (m, 2H), 4.14 (q, J = 7.1 Hz, 2H), 3.08 (dd, J = 8.1 , 2.9 Hz, 1 H), 2.75 (td, J = 12.6, 5.0 Hz, 1 H), 2.51 (td, J = 12.6, 4.6 Hz, 1 H), 2.42 - 2.31 (m, 5H), 2.31 - 2.16 (m, 4H), 1.77 - 1.63 (m, 5H), 1.63 - 1.51 (m, 1 H), 1.35 - 1.19 (m, 3H), 0.97 (s, 9H), 0.67 (t, J = 7.3 Hz, 6H).13C NMR (126 MHz, CDCI3) 5 173.37, 169.98, 166.58, 163.58, 146.35, 139.33, 130.61 , 129.31 , 128.27, 127.39, 127.33, 119.22, 116.03, 88.54, 74.57, 65.30, 60.28, 53.14, 36.21 , 33.93, 30.20, 28.36, 27.46, 26.43, 23.56, 21.57, 21.51 , 14.27, 8.52.(120) (122)Methyl 4-(3-(5-(3-(benzyloxy)-4,4-dimethylpentyl)-4-methyl-6-oxo-1,6-dihydropyrimidin-2- yl)pentan- 3-yl)benzoate (122)

[0220] Dissolved 120 (200 mg, 0.37 mmol, 1 equiv.) in DMSO (1.5 mL) then added methanol (1.5 mL). Added palladium acetate (9 mg, 0.037 mmol, 0.1 equiv) and 1 ,3-Bis(diphenylphosphino)propane (15 mg, 0.037 mmol, 0.1 equiv.). Let stir at room temperature for 5 minutes then added triethylamine (130 pL, 0.93 mmol, 2.5 equiv.). The reaction vessel was evacuated (carefully) until solvent began to boil and then filled with 2 atm of CO and sealed. The stirred reaction mixture was heated to 90°C for 24 hours. The reaction was cooled to room temperature and then quenched with 1 M HCI (aq.) (10 mL). The mixture was extracted with DCM (3x 15 mL) and then the combined organic layers were washed with brine (10 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. Purified by FCC (20% ethyl acetate in hexanes) to give 122 as a white solid (91% yield, 175 mg). IR (thin film) v 2964, 2870, 1723, 1637 cm- 1.1H NMR (500 MHz, CDCI3) 69.67 (br. S, 1 H), 8.04 - 7.93 (m, 2H), 7.44 (d, J = 7.1 Hz, 2H), 7.37 (dd, J = 8.4, 6.7 Hz, 2H), 7.32 - 7.29 (m, 1 H), 7.27 (d, J = 2.0 Hz, 2H), 4.72 (q, J = 11 .4 Hz, 2H), 3.92 (s, 3H), 3.13 (dd, J = 8.8, 2.7 Hz, 1 H), 2.59 (dtd, J = 47.9, 12.6, 4.9 Hz, 2H), 2.35 (s, 3H), 2.22 (ddt, J = 14.7, 7.4, 3.8 Hz, 2H), 2.14 (dd, J = 14.4, 7.4 Hz, 2H), 1.77 (dddd, J = 14.2, 11.7, 4.9, 2.7 Hz, 1H), 1.60 - 1.52 (m, 1 H), 0.98 (s, 9H), 0.74 - 0.60 (m, 6H).13C NMR (126 MHz, CDCI3) 6 166.67, 163.27, 159.86, 159.67, 148.26, 139.42, 129.74, 128.90, 128.27, 127.62, 127.43, 127.28, 122.94, 88.56, 74.84, 52.28, 52.13, 36.26, 29.77, 27.02, 26.50, 23.74, 21.73, 7.96. HRMS (ESI+) calculated for C32H42O4N2 [M+Na]+: 541.3037, found 541.3015.Methyl 4-(3-(5-(3-(benzyloxy)-4,4-dimethylpentyl)-4-chloro-6-methylpyrimidin-2-yl)pentan-3- yl)benzoate (123)

[0221] To a solution of 122 (218 mg, 0.42 mmol, 1 equiv.) in anhydrous toluene (1 mL), added POCI3 (80 pL, 0.84 mmol, 2 equiv.) dropwise at room temperature. Heated to reflux for 2 hours. Cooled to room temperature and quenched the reaction mixture by pouring into ice water (25 mL). Adjusted the pH to 5 using 3M NaOH (aq.). Extracted with DCM (3x 15 mL). Dried combined organic layers over anhydrous sodium sulfate. Purified via FCC (10% ethyl acetate in hexanes) to give 123 as a yellow oil (221 mg, 98% yield). IR (thin film) v 2964, 2875, 1721 , 1561 , 1519, 1434 cm’1.1H NMR (500 MHz, CDCI3) 5 7.96 - 7.90 (m, 2H), 7.46 - 7.34 (m, 4H), 7.34 - 7.29 (m, 3H), 4.81 - 4.59 (m, 2H), 3.90 (s, 3H), 3.14 (dd, J = 8.7, 2.7 Hz, 1 H), 2.91 (td, J = 13.0, 4.8 Hz, 1 H), 2.66 (td, J = 12.9, 4.4 Hz, 1 H), 2.49 - 2.38 (m, 5H), 2.31 (dt, J = 14.0, 7.2 Hz, 2H), 1.81 (dddd, J = 13.8, 12.6, 4.5, 2.7 Hz, 1 H), 1.67 - 1.54 (m, 1 H), 0.99 (s, 9H), 0.67 (t, J = 7.3 Hz, 6H).13C NMR (126 MHz, CDCI3) 5 171.63, 167.16, 166.55, 160.38, 151.90, 139.06, 129.14, 128.62, 128.35, 127.57, 127.49, 88.56, 75.18, 53.81 , 51.94, 36.36, 29.74, 27.45, 27.16, 26.47, 22.51 , 8.38. HRMS (ESI+) calculated for C32H41O3N2CI [M+Na]+: 559.2698, found 559.2685.Methyl 4-(3-(5-(3-hydroxy-4,4-dimethylpentyl)-4-methylpyrimidin-2-yl)pentan-3-yl)benzoate (124)

[0222] To a solution of 123 (50 mg, 0.09 mmol, 1 equiv.) in a 1 :1 mixture of methanol-ethyl acetate (2 mL) added palladium hydroxide on carbon (10 mg, 20% Pd on carbon) and triethylamine (26 pL, 0.18 mmol, 2 equiv.). Evacuated the reaction flask and filled with argon (3x) then established an atmosphere of hydrogen using a balloon and a vent to relieve the round-bottom flask of all argon. Vigorously stirred the solution for 20 hours before filtering over a pad of celite. Concentrated the filtrate under reduced pressure and purified via column chromatography (25% ethyl acetate in hexanes) to give 124 as a white solid (8.5 mg, 22% yield). IR (thin film) v 3477, 2962, 2875, 1721 , 1549, 1434cm’1 1H NMR (500 MHz, CDCI3) 6 8.38 (s, 1 H), 7.93 (d, J = 8.7 Hz, 2H), 7.37 - 7.14 (m, 2H), 3.89 (s, 3H), 3.23 (dd, J = 10.7, 1.8 Hz, 1 H), 2.91 (ddd, J = 14.9, 10.4, 4.7 Hz, 1 H), 2.57 (ddd, J = 14.3, 10.2, 6.5 Hz, 1 H), 2.49 (s, 3H), 2.45 (ddd, J = 13.7, 7.4, 1.4 Hz, 2H), 2.31 (dq, J = 14.4, 7.3 Hz, 2H), 1.77 (dddd, J = 13.8, 10.5, 6.5, 1.8 Hz, 1 H), 1.54 (dtd, J = 13.7, 10.4, 4.8 Hz, 1 H), 0.91 (s, 9H), 0.66 (t, J = 7.4 Hz, 6H).13C NMR (126 MHz, CDCI3) 6 171.70, 167.22, 164.66, 155.92, 152.85, 129.61 , 129.12, 127.51 , 127.38, 79.40, 53.93, 51.91 , 35.02, 31.18, 27.79, 27.08, 25.59, 21.95, 8.42. HRMS (ESI+) calculated for C25H36O3N2 [M+Na]+: 435.2618, found 435.2605.4-(3-(5-(3-(Benzyloxy)-4,4-dimethylpentyl)-4-methylpyrimidin-2-yl)pentan-3-yl)benzoic acid (124b)

[0223] To a solution of 123 (50 mg, 0.09 mmol, 1 equiv.) in a 1 :1 mixture of methanol-ethyl acetate (2 mL) added palladium hydroxide on carbon (10 mg, 20% Pd on carbon) and triethylamine (26 pL, 0.18 mmol, 2 equiv.). Evacuated the reaction flask and filled with argon (3x) then established an atmosphere of hydrogen using a balloon and a vent to relieve the round-bottom flask of all argon. Vigorously stirred the solution for 20 hours before filtering over a pad of celite. Concentrated the filtrate under reduced pressure and purified via column chromatography (25% ethyl acetate in hexanes) to give 124b as a gummy yellow solid (30 mg, 65% yield).1H NMR (500 MHz, CDCI3) 68.34 (s, 1 H), 7.93 (d, J = 8.6 Hz, 2H), 7.43 - 7.34 (m, 4H), 7.33 - 7.26 (m, 3H), 4.88 - 4.51 (m, 2H), 3.89 (s, 3H), 3.11 (dd, J= 9.2, 2.7 Hz, 1 H), 2.79 (ddd, J = 14.1 , 11.4, 5.1 Hz, 1 H), 2.55 - 2.38 (m, 6H), 2.31 (dq, J = 14.4, 7.3 Hz, 2H), 1.85 - 1.65 (m, 2H), 0.99 (s, 9H), 0.66 (t, J = 7.4 Hz, 6H).13C NMR (126 MHz, CDCI3) 6 171.15, 167.21 , 164.58, 155.72, 152.84, 138.98, 129.73, 129.12, 128.40, 127.55, 127.50, 127.47, 127.38, 88.23, 75.41 , 53.92, 51.91 , 36.33, 31.29, 27.79, 27.47, 26.48, 21.93, 8.42. HRMS (APCI+) calculated for C32H42N2O3 [M+H]+: 503.3268, found 503.3285.4-(3-(5-(3-Hydroxy-4,4-dimethylpentyl)-4-methylpyrimidin-2-yl)pentan-3-yl)benzoic acid (125)

[0224] LiOH H2O (11 mg, 0.26 mmol, 6.5 equiv.) was added in one portion to a vigorously stirring solution of 124 (17.9 mg, 0.04 mmol, 1 equiv.) in methanol and water (1 :1 , 0.5 mL) under air. Stirred for 6 hours at room temperature then quenched with 1 M HCI (aq.) until the pH <1 . Extracted with ethyl acetate (3x 5 mL), the washed the combined organic layers with brine (5 mL), dried over anhydrous sodium sulfate, and then concentrated the organic layers under reduced pressure to give 17 mg (98% yield) of 125 as white solid which was used without further purification. IR (thin film) v 3441 , 2958, 2848, 1686, 1641 cm’1.1H NMR (500 MHz, CDCI3) 58.43 (s, 1 H), 7.93 (d, J= 8.5 Hz, 2H), 7.29 (dd, J = 6.6 Hz, 2H), 3.23 (dd, J = 10.7, 1.8 Hz, 1 H), 2.92 (ddd, J = 14.7, 10.3, 4.8 Hz, 1 H), 2.60 (ddd, J = 14.3, 10.0, 6.5 Hz, 1 H), 2.54 - 2.39 (m, 5H), 2.33 (td, J = 13.3, 6.1 Hz, 2H), 1.86 - 1.71 (m, 1H), 1.66 - 1.52 (m, 1 H), 0.91 (s, 9H), 0.66 (t, J = 7.4 Hz, 6H).13C NMR (126 MHz, CDCI3) 5 171.56, 171.23, 164.95, 155.89, 153.30, 129.81 , 129.70, 127.67, 126.87, 79.38, 54.09, 35.03, 31.05, 27.92, 27.09, 25.60, 21.93, 8.41. HRMS (ESI+) calculated for C24H34N2O3 [M+H]+: 399.2642, found 399.2658.(125) (128)Methyl 6-(4-(3-(5-(3-hydroxy-4,4-dimethylpentyl)-4-methylpyrimidin-2-yl)pentan-3- yl)benzamido)hexanoate (128)

[0225] EDC HCI (10 mg, 0.05 mmol, 1.1 equiv.), DMAP(1 mg, 0.004 mmol, 0.1 equiv.), and Et3N (13 pL, 0.09 mmol, 2 equiv.) were then added in that order to a stirring solution of 125 (18 mg, 0.045 mmol, 1 equiv.) and methyl 6-aminohexanoate hydrochloride (12 mg, 0.068 mmol, 1.5 equiv.) in anhydrous DCM (2.25 mL) at room temperature overnight. Once complete as determined by TLC analysis, the reaction mixture was quenched with 2.5 mL of a 1 M HCI (aq.) solution and extractedwith ethyl acetate (3x 15 mL) and followed by a 5 mL wash with a brine solution. The organic fractions were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by FCC (5% methanol in dichloromethane) to give 128 as an orange solid (18.4 mg, 81 % yield). IR (thin film) v 3346, 2952, 2871 , 1735, 1638, 1544, 1423 cm’1.1H NMR (500 MHz, CDCI3) 6 8.37 (s, 1 H), 7.65 (d, J = 8.5 Hz, 2H), 7.28 (d, J = 8.6 Hz, 2H), 6.14 (t, J = 5.9 Hz, 1 H), 3.67 (s, 3H), 3.45 (td, J = 7.0, 5.8 Hz, 2H), 3.23 (dd, J = 10.6, 1.8 Hz, 1 H), 2.90 (ddd, J = 14.8, 10.5, 4.7 Hz, 1 H), 2.57 (ddd, J = 14.3, 10.2, 6.4 Hz, 1 H), 2.49 - 2.40 (m, 5H), 2.37 - 2.26 (m, 4H), 1.76 (dddd, J = 13.8, 10.5, 6.4, 1.8 Hz, 1 H), 1.71 - 1.65 (m, 2H), 1.61 (q, J = 7.4 Hz, 2H), 1.56 - 1.50 (m, 1 H), 1.45 - 1.37 (m, 2H), 1.27 (d, J = 1.9 Hz, 1 H), 0.91 (s, 9H), 0.65 (t, J = 7.4 Hz, 6H).13C NMR (126 MHz, CDCI3) 6 174.07, 171.79, 167.60, 164.63, 155.90, 151.06, 131.93, 129.57, 127.65, 126.37, 79.40, 53.74, 51.52, 39.64, 35.03, 33.87, 31.20, 29.35, 27.77, 27.09, 26.40, 25.60, 24.47, 21.96, 8.42. HRMS (ESI+) calculated for C31 H47N3O4 [M+Na]+: 548.3459, found 548.3450.Methyl 5-(4-(3-(5-(3-hydroxy-4,4-dimethylpentyl)-4-methylpyrimidin-2-yl)pentan-3- yl)benzamido)pentanoate (127)

[0226] EDC HCI (12 mg, 0.06 mmol, 1.1 equiv.), DMAP(1 mg, 0.005 mmol, 0.1 equiv.), and Et3N (14 pL, 0.1 mmol, 2 equiv.) were then added in that order to a stirring solution of 125 (20 mg, 0.05 mmol, 1 equiv.) and methyl 5-aminovalerate hydrochloride (13 mg, 0.075 mmol, 1.5 equiv.) in anhydrous DCM (2.5 mL) at room temperature overnight. Once complete as determined by TLC analysis, the reaction mixture was quenched with 2.5 mL of a 1 M HCI (aq.) solution and extracted with DCM (3x 15 mL) and followed by a 5-mL wash with a brine solution. The organic fractions were dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by FCC (2- 4% methanol in dichloromethane) to give 127 as an orange solid (22 mg, 72% yield). IR (thin film) v 3321 , 2959, 2873, 1738, 1639, 1548, 1434 cm’1.1H NMR (500 MHz, CDCI3) 6 8.38 (s, 1 H), 7.66 (d, J = 8.0 Hz, 2H), 7.32 - 7.20 (m, 2H), 6.23 (t, J = 5.8 Hz, 1 H), 3.69 (d, J = 2.1 Hz, 3H), 3.54 - 3.41 (m, 2H), 3.29 - 3.19 (m, 1 H), 2.90 (d, J = 4.3 Hz, 1 H), 2.57 (ddd, J= 14.2, 10.1 , 6.4 Hz, 1 H), 2.49 (s, 3H), 2.45 (dd, J = 14.0, 7.2 Hz, 2H), 2.38 (t, J = 7.1 Hz, 2H), 2.31 (dq, J = 14.2, 7.3 Hz, 2H), 1.83 - 1.69 (m, 3H), 1.65 (p, J= 7.1 Hz, 2H), 1.56 (td, J = 9.9, 4.9 Hz, 1 H), 1.32 - 1.23 (m, 1H), 0.91 (s, 9H), 0.66(t, J = 7.3 Hz, 6H).13C NMR (126 MHz, CDCI3) 6 174.04, 171.79, 167.62, 164.65, 155.90, 151.09, 131.83, 129.57, 127.67, 126.38, 79.42, 53.75, 51.62, 39.37, 35.03, 33.48, 31.19, 29.08, 27.76, 27.09, 25.59, 22.04, 21.96, 8.42. HRMS (APCI+) calculated forC30H45N3O4 [M+H]+: 512.3483, found 512.3484.Ethyl 4-(4-(3-(5-(3-hydroxy-4,4-dimethylpentyl)-4-methylpyrimidin-2-yl)pentan-3- yl)benzamido)butanoate (126)

[0227] EDC HCI (14 mg, 0.075 mmol, 1.1 equiv.), DMAP(1 mg, 0.006 mmol, 0.1 equiv.), and Et3N (18 pL, 0.13 mmol, 2 equiv.) were then added in that order to a stirring solution of acid 125 (25 mg, 0.063 mmol, 1 equiv.) and ethyl 4-aminobutyrate (16 mg, 0.094 mmol, 1.5 equiv.) in anhydrous DCM (3.15 mL) at room temperature overnight. Once complete as determined by TLC analysis, the reaction mixture was quenched with 3 mL of a 1 M HCI (aq.) solution and extracted with DCM (3x 15 mL) and followed by a 5-mL wash with a brine solution. The organic fractions were dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by FCC (2-4% methanol in dichloromethane) to give 126 as an orange solid (21 mg, 67% yield). I R (thin film) v 3321 , 2963, 2874, 1734, 1638, 1545, 1503, 1425 cm’1.1H NMR (500 MHz, CDCI3) 6 8.37 (s, 1H), 7.66 (d, J = 8.0 Hz, 2H), 7.28 (d, J = 8.4 Hz, 2H), 6.47 (q, J = 6.1 Hz, 1 H), 4.13 (p, J = 7.1 Hz, 2H), 3.49 (q, J = 6.4 Hz, 2H), 3.23 (dd, J = 10.6, 1.8 Hz, 1 H), 2.90 (ddd, J = 14.8, 10.5, 4.7 Hz, 1 H), 2.65 - 2.51 (m, 1 H), 2.51 - 2.34 (m, 7H), 2.30 (dd, J = 14.0, 7.2 Hz, 2H), 1.97 - 1.91 (m, 2H), 1.76 (dddd, J = 12.7, 10.5, 6.6, 1.8 Hz, 1 H), 1.60 - 1.48 (m, 1 H), 1.26 (dt, J = 15.8, 7.1 Hz, 4H), 0.91 (s, 9H), 0.65 (t, J = 7.3 Hz, 6H).13C NMR (126 MHz, CDCI3) 6 173.80, 171.78, 167.57, 164.64, 155.89, 151.14, 131.62, 129.58, 127.66, 126.40, 79.40, 60.64, 53.75, 39.57, 35.03, 31.99, 31.20, 27.76, 27.09, 25.60, 24.50, 21.95, 14.19, 8.42. HRMS (APCH-) calculated for C30H45N3O4 [M+H]+: 512.3483, found 512.3488.(128) (50)4-(3-(5-(3-Hydroxy-4,4-dimethylpentyl)-4-methylpyrimidin-2-yl)pentan-3-yl)-N-(6- (hydroxyamino)-6- oxohexyl)benzamide (50) (AC-451)

[0228] Hydroxylamine (1 ml, 16.1 mmol, aq. 50%wt solution, 500 equiv.), followed by 3M KOH (aq.)(0.1 mL, 0.226 mmol, 7 equiv.) were added in that order to a vigorously stirring solution of 128 (17 mg, 0.032 mmol, 1 equiv.) at 0°C in a 1 :1 MeOH / THF mixture (4 mL) under air. The reaction was left to warm slowly to room temperature overnight. After 24 hours, removed volatiles, added 3mL water and adjusted to pH = 7 with 1M HCI (aq.), then extracted with HPLC grade ethyl acetate (3 mL, 3x). Removed volatiles under reduced pressure and purified by reversed-phase chromatography on octadecyl-functionalized silica gel using a gradient of 60-95% MeOH:H2O over a period of 20 minutes as eluent to provide AC-451 (50)(16 mg, 95% yield) as a fine white powder after lyophilization. IR (thin film) v 3190, 2934, 1631 , 1546, 1426 cm’1 1H NMR (500 MHz, DMSO) 5 10.29 (s, 1 H), 8.62 (s, 1 H), 8.41 (s, 1 H), 8.29 (d, J = 5.8 Hz, 1 H), 7.75 - 7.45 (m, 2H), 7.16 (d, J = 8.4 Hz, 2H), 4.51 (d, J = 6.3 Hz, 1 H), 3.19 (q, J = 6.7 Hz, 2H), 3.01 - 2.91 (m, 1 H), 2.78 (d, J = 9.8 Hz, 1 H), 2.39 (s, 4H), 2.36 - 2.28 (m, 2H), 2.28 - 2.17 (m, 2H), 1.92 (t, J = 7.4 Hz, 2H), 1.65 (t, J = 10.4 Hz, 1 H), 1.47 (h, J = 7.8 Hz, 4H), 1.35 (qd, J = 11.1 , 4.4 Hz, 1 H), 1.27 - 1.20 (m, 2H), 0.79 (s, 9H), 0.56 (t, J = 7.3 Hz, 6H).13C NMR (126 MHz, DMSO) 5 171.26, 169.45, 166.50, 164.71 , 156.32, 150.53, 132.50, 130.60, 127.35, 127.11 , 77.61 , 53.62, 39.42, 35.26, 32.69, 31.55, 29.37, 27.69, 26.90, 26.54, 26.35, 25.36, 22.15, 8.83.HRMS (ESI+) calculated for C30H46N4O4 [M+H]+: 527.3592, found 527.3608. Purity >96% by LC-MS, tr = 11.13 minutes.(127) (44)4-(3-(5-(3-Hydroxy-4,4-dimethylpentyl)-4-methylpyrimidin-2-yl)pentan-3-yl)-N-(5- (hydroxyamino)-5- oxopentyl) benzamide (44) (AC-454)

[0229] Hydroxylamine (1.1 ml, 17.6 mmol, aq. 50%wt solution, 500 equiv.), followed by 3M KOH (aq.) (0.1 mL, 0.25 mmol, 7 equiv.) were added in that order to a vigorously stirring solution of 127 (18 mg, 0.035 mmol, 1 equiv.) at 0°C in a 1 : 1 MeOH / THF mixture (4.4 mL) under air. The reaction was left to warm slowly to room temperature overnight. After 24h, removed volatiles and added 3mL water and adjusted to pH = 7 with 1M HCI (aq.), then extracted with HPLC grade ethyl acetate (3 mL, 3x). Removed volatiles under reduced pressure and purified by reversed-phase chromatography on octadecyl-functionalized silica gel using a gradient of 60-95% MeOH:H2O over a period of 20 minutes as eluent to provide AC-454 (44) (12.4 mg, 69% yield) as a fine white powder after lyophilization. IR (thin film) v 3243, 2960, 2869, 1634, 1541 , 1424 cm’1.1H NMR (500 MHz, DMSO) 5 10.31 (s, 1 H), 8.63 (s, 1 H), 8.40 (s, 1 H), 8.32 (t, J = 5.7 Hz, 1 H), 7.66 (d, J = 8.2 Hz, 2H), 7.17 (d, J = 8.1 Hz, 2H), 4.51 (d, J = 6.2 Hz, 1 H), 3.19 (q, J = 6.4 Hz, 2H), 3.02 - 2.91 (m, 1 H), 2.79 (td, J = 10.1 , 5.1 Hz, 1 H), 2.39 (s, 4H), 2.37 - 2.28 (m, 2H), 2.28 - 2.17 (m, 2H), 1.94 (t, J = 7.0 Hz, 2H), 1.64 (q, J = 9.9 Hz, 1 H), 1.49 (dd, J = 16.1 , 8.5 Hz, 4H), 1.41 - 1.31 (m, 1 H), 0.79 (s, 9H), 0.56 (t, J = 7.2 Hz, 6H).13C NMR (126 MHz, DMSO) 5 171.26, 169.43, 166.53, 164.72, 156.32, 150.55, 132.47, 130.60, 127.36, 127.10, 77.61 , 53.63, 39.26, 35.26, 32.45, 31.54, 29.24, 27.69, 26.90, 26.35, 23.16, 22.15, 8.83. HRMS (ESI+) calculated for C39H44N4O4 [M+H]+: 513.3435, found 513.3432. Purity >98 % by LC-MS, tr = 10.82 minutes.(126) (43)4-(3-(5-(3-Hydroxy-4,4-dimethylpentyl)-4-methylpyrimidin-2-yl)pentan-3-yl)-N-(4- (hydroxyamino)-4-oxobutyl) benzamide (43) (AC-455)

[0230] Hydroxylamine (1.3 mL, 20.5 mmol, aq. 50%wt solution, 500 equiv.), followed by 3M KOH (aq.) (0.1 mL, 0.287 mmol, 7 equiv.) were added in that order to a vigorously stirring solution of 126 (21 mg, 0.041 mmol, 1 equiv.) at 0°C in a 1 :1 MeOH / THF mixture (5.1 mL) under air. The reaction was left to warm slowly to room temperature overnight. After 24 hours, removed volatiles and added 3mL water and adjusted to pH = 7 with 1M HCI (aq.), then extracted with HPLC grade ethyl acetate (3 mL, 3x). Removed volatiles under reduced pressure and purified by reversed- phase chromatography on octadecyl-functionalized silica gel using a gradient of 60-95% MeOH:H2O over a period of 20 minutes as eluent to provide AC-455 (43) (14.1 mg, 69% yield) as a fine white powder after lyophilization. IR (thin film) v 3214, 2962, 2873, 1633, 1541 , 1423 cm- 1.1H NMR (500 MHz, DMSO) 5 10.34 (s, 1 H), 8.66 (s, 1 H), 8.41 (s, 1 H), 8.35 (t, J = 5.9 Hz, 1 H), 7.77 - 7.56 (m, 2H), 7.31 - 7.11 (m, 2H), 4.51 (d, J = 6.2 Hz, 1 H), 3.20 (d, J = 5.9 Hz, 2H), 2.96 (t, J = 8.1 Hz, 1 H), 2.78 (d, J = 13.8 Hz, 1 H), 2.41 - 2.28 (m, 6H), 2.28 - 2.17 (m, 2H), 1.97 (t, J = 7.4 Hz, 2H), 1.77 - 1 .58 (m, 3H), 1 .35 (d, J = 9.4 Hz, 1 H), 0.79 (s, 9H), 0.56 (t, J = 7.2 Hz, 6H).13C NMR (126 MHz, DMSO) 5 171.25, 169.30, 166.60, 164.72, 156.32, 150.61 , 132.38, 130.61 , 127.37, 127.13, 77.61 , 53.63, 39.26, 35.26, 31.55, 30.44, 27.69, 26.90, 26.35, 25.79, 22.15, 8.83. HRMS (ESI+) calculated for C38H42N4O4 [M+H]+: 499.3275, found 499.3279. Purity >99 % by LC-MS, tr = 10.69 minutes.(67) (152)2-(4-(Benzyloxy)phenyl)-2-ethylbutanamide (152)100°C " Jays775

[0231] In a screw capped vial, added freshly powdered potassium hydroxide (430 mg, 7.65 mmol, 2 equiv.), 8 mL of dry f-butanol (dried over 3 molecular sieves), and 67 (1.07g, 3.8 mmol, 1 equiv.). Sealed the vial and stirred at 100°C for 5 days. Diluted with dichloromethane (8 mL) and deionized.

[0232] The synthesis of these hybrids began with double alkylation of 4-(benzyloxy)phenylacetonitrile using LDA and ethyl iodide to afford 9 in 88% yield (Scheme above). The nitrile in 9 was hydrolyzed to the corresponding amide 10 under basic conditions in 73% yield, and the necessary thioamide 11 was then generated by treatment with Lawesson’s reagent in 25% yield. Preparation of bromoketone 17a began with alkylation of the dimethyl hydrazone of pinacolone with 4-bromo-2-methyl -3 -butene to produce ketone 13 in 37% yield. Reduction of the ketone with NaBEL followed by TBS protection and ozonolysis afforded 16 in 32% over three steps. Finally, a-halogenation with NBS afforded the key bromoketone 17a along with its corresponding 1 -bromo analog 17b. The latter was inseparable from 17b but could be easily removed after selective reaction with 4-chlorobenzenethiol.

[0233] The thiazole was constructed as planned by condensation of 11 with 17a to afford 18 in 67% yield. Elaboration to the hybrids was achieved by hydrogenolysis of the benzyl group, in 62% yield, followed by alkylation of the resulting phenol with appropriate length bromoesters followed by condensation with hydroxylamine. This provided hybrids 7a (AC-340), 7b (AC-339) and 7c (AC-341), with linking chain lengths of 3-, 4- and 5-carbons, in yields of 26- 49% over the final three steps2-(4-(Benzyloxy)phenyl)-2-ethylbutanenitrile (9)

[0234] To a solution of diisopropylamine (3.9 mL, 27.7 mmol, 2.8 equiv.) in anhydrous THF (52 mL) at - 78 °C was added n-butyl lithium (10.3 mL, 2.5 M in hexanes). Let stir for 1 minute. A solution of 2-(4-(benzyloxy)phenyl)acetonitrile (8)1(2.2 g, 9.89 mmol, 1 equiv.) in 18 mL of anhydrous THF was added dropwise via cannula. Stirred at -78 °C for 15 minutes. Ethyl iodide (2.2 mL, 27.7 mmol, 2.8 equiv.) in 35 mL anhydrous THF was added dropwise to the reaction mixture via cannula. Stirred for 15 minutes at -78 °C, then 1 hour at 0 °C. Monitored reaction by TLC. Quenched with 1 M NH4CI (aq.)(50 mL) and dissolved in ethyl acetate (50 mL). Washed organic layer with brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure. Purified via FCC (15:1 hexanes : ethyl acetate) to give 9 as a white solid (2.4 g, 88% yield). Rf= 0.35 (10: 1 hexanes: ethyl acetate). IR (thin film) v 2973, 2936, 2877, 2235 cm’1.1H NMR (500 MHz, CDCI3) 57.48 - 7.32 (m, 5H), 7.32 - 7.28 (m, 2H), 7.02 - 6.96 (m, 2H), 5.08 (s, 2H), 2.19 - 1.97 (m, 2H), 1.97 - 1.77 (m, 2H), 0.92 (t, J = 7.4 Hz, 6H).13C NMR (126 MHz, CDCI3) 5 158.13, 136.79, 130.32, 128.64, 128.09, 127.56, 127.30, 122.54, 114.98, 70.12, 49.12, 33.86, 9.70. HRMS (ESI) calculated for C19H21NO [M+Na]+: 302.1521 , found 302.1515.1. Ellingboe, J. W.; Alessi, T. R.; Dolak, T. M.; Nguyen, T. T.; Tomer, J. D.; Guzzo, F.; Bagli, J. F.; McCaleb, M. L. J. Med. Chem. 1992, 35 (7), 1176-1183.2,2,7-Trimethyloct-7-en-3-one (13)

[0235] To a solution of 2-butanone, 3,3-dimethyl-dimethylhydrazone (12)1(8.7 g, 61 mmol, 1 equiv.) in THF (100 mL) was added dropwise n-BuLi (26.9 mL, 2.5 M solution in hexanes) at 0 °C. The mixture was stirred for 30 min, and 4-Bromo-2-methylbut-1-ene2(10 g, 67 mmol, 1.05 equiv.) was added dropwise. The mixture was stirred at room temperature for 10 h, quenched by addition of water (75 mL), and extracted with EtOAc (75 mL, 2x).The organic phase was concentrated, after which acetone (163 mL) and acidic resin (Amberlyte IR 120, 16.6 g) were added. The mixture was stirred for 12 h, concentrated, and purified by FCC ( 5% ethyl acetate in hexanes) to give 13 as a yellow oil. (37% yield, 3.8g). Rf = 0.45 (5:95, ethyl acetate to hexanes). IR (thin film) v 2966, 2871 , 1705, 1477, 1366 cm’1.1H NMR (500 MHz, CDCI3) 4.72 (s, 1 H), 4.68 (s,1 H), 2.48 (t, J =, 2H), 2.01 (t, J =, 2H), 1.71 (m, 5H), 1.14 (s, 9H).13C NMR (126 MHz, CDCI3) 5 219, 145, 110, 44, 37, 35, 26, 22.1 , 21.5. HRMS (ESI) calculated for CnH2oO[M+Na]+; 191.1406, found 191.1403.1. Hsu, J. L.; Fang, J. M. J. Org. Chem. 2001 , 66 (25), 8573-8584.2. Berkowitz, W. F.; Wu, Y.; Topics, T. A. C. J. Org. Chem. 1997, 3263 (27), 1536-15392,2,7-Trimethyloct-7-en-3-ol (14)

[0236] To a solution of 13 (559 mg, 3.3 mmol, 1 equiv.) in dry methanol (3.3 mL, 1.0 M) at 0 °C was slowly added NaBF (185 mg, 4.9 mmol, 1.5 equiv.). The resulting mixture was warmed to room temperature and stirred for 1 h. Water (5 mL) was added and the product extracted with DCM (10 mL). The combined organic layers were washed with brine (10 mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by chromatography (10% ethyl acetate in hexanes) to give 14 as a colourless oil (492 mg, 75% yield). Rf= 0.32 (10% Ethyl acetate in hexanes). IR (thin film) v 3405, 2952, 2868, 1462, 1363 cm’1.1H NMR (300 MHz, CDCI3) 4.70 (s, 1 H), 4.68 (s, 1 H), 3.19 (dd, J = , 1 H) , 2.07-2.02 (m, 2H), 1.82- 1.67 (m, 4H), 1.59-1.38 (m, 3H), 1.30-1.22 (m, 1 H), 0.88 (s, 9H).13C NMR (126 MHz, CDCI3) 6 145.92, 109.89, 79.88, 37.78, 34.97, 31.10, 25.69, 25.05, 22.38. HRMS (ESI) calculated for CnH22O[M+Na]+; 193.1563, found 193.1568.Tert-butyldimethyl((2,2,7-trimethyloct-7-en-3-yl)oxy)silane (15)

[0237] To a solution of 2,2,7-trimethyloct-7-en-3-ol (310 mg, 1.8 mmol, 1 equiv.) in DMF (0.92 mL, 2 M) at 0 °C, added imidazole (313 mg, 4.6 mmol, 2.55 equiv.) followed by TBSCI (415 mg, 2.7 mmol, 1.5 equiv.). Stirred for 3 days at room temperature. Added water (10 mL) and extracted with DCM (10 mL, 3x), washed combined organic layers with brine (15 mL) and dried over anhydrous sodium sulfate. Purified via FCC (2% ethyl acetate in hexanes) to give Tert- butyldimethyl((2,2,7-trimethyloct-7-en-3-yl)oxy)silane as a colourless oil (322 mg, 63% yield). Rf= 0.72 (5% ethyl acetate in hexanes). IR (thin film) v 2953, 2867, 1462, 1363 cm’1.1H NMR (500 MHz, CDCI3) 4.72 (s, 1 H), 4.69 (s, 1 H), 3.25 (dd, J =, 1 H), 2.01-1.98 (m, 2H), 1.74 (s, 3H), 1.69 - 1.5 (m, 2H), 1.40 - 1.27 (m, 2H), 0.92 (s, 9H), 0.88 (s, 9H), 0.08 (3H), 0.06 (3H).13C NMR (126 MHz, CDCI3) 6 146, 109, 80, 38.3, 35.8, 67 33.1 , 26.5, 26.1 , 25.7, 22.4, 18.43, 3.3, 3.9. HRMS (ESI) calculated for Ci7H36OSi [M+Na]+: 307.2428, found 307.2430.6-((tert-Butyldimethylsilyl)oxy)-7,7-dimethyloctan-2-one (16)

[0238] A stream of ozone was bubbled through a solution of tert-butyldimethyl((2,2,7- trimethyloct-7-en-3-yl)oxy)silane (100 mg, 0.35 mmol, 1 equiv.) in 6 mL dry dichloromethane at - 78 °C until the solution turned blue. Excess ozone was removed by passing a stream of nitrogen through the solution. Triphenylphosphine (102 mg, 0.39 mmol, 1.05 equiv.) was added slowly,and the solution warmed to room temperature overnight. The solution was concentrated in vacuo and the residue purified by FCC (2% diethyl ether in hexanes) to give 6-((tert- Butyldimethylsilyl)oxy)-7,7-dimethyloctan-2-one as a colourless oil (79 mg, 79% yield). Rf = 0.35 (5% ethyl acetate in hexanes). IR (thin film) v 2954, 2856, 1718, 1360 cm’1.1H NMR (500 MHz, CDCI3) 6 3.23 (dd, J = 6.9, 3.1 Hz, 1 H), 2.41 (td, J = 7.1 , 1.7 Hz, 2H), 2.15 (s, 3H), 1.77 (ddd, J = 12.6, 6.9, 2.0 Hz, 1 H), 1.53 (ttd, J = 11.0, 5.1 , 2.5 Hz, 2H), 1.38 - 1.23 (m, 2H), 0.92 (s, 9H), 0.86 (s, 9H), 0.07 (d, J = 9.5 Hz, 6H).13C NMR (126 MHz, CDCI3) 6208.88, 80.42, 44.25, 35.85, 33.04, 29.80, 26.44, 26.15, 22.06, 18.39, -3.40, -3.98. HRMS (ESI) calculated for Ci6H34O2Si [M+Na]+: 309.2226, found 309.2224.2-(4-(Benzyloxy)phenyl)-2-ethylbutanamide (10)

[0239] In a screw capped vial, added freshly powdered potassium hydroxide (430 mg, 7.65 mmol, 2 equiv.), 8 mL of dry t-butanol (dried over 3 molecular sieves), and 2-(4- (Benzyloxy)phenyl)-2-ethylbutanenitrile (1.07g, 3.8 mmol, 1 equiv.). Sealed the vial and stirred at 100 °C for 5 days. Diluted with dichloromethane (8 mL) and deionized 85 water (10 mL). Extracted the aqueous layer with DCM (8 mL, 3x). Dried the organic layer over anhydrous sodium sulfate, concentrated under reduced pressure and purified via FCC (1 :1 ethyl acetate : hexanes) to give 2-(4-(Benzyloxy)phenyl)-2-ethylbutanamide as a white solid (0.83 g, 73% yield). Rf = 0.36 (1 :1 hexanes : ethyl acetate). IR (thin film) v 3468, 3156, 2968, 2934, 2877, 1667, 1509, 1455 cm’1.1H NMR (500 MHz, CDCI3) 6 7.50 - 7.31 (m, 5H), 7.27 - 7.22 (m, 2H), 7.03 - 6.89 (m, 2H), 5.19 (d, J = 19.5 Hz, 2H), 5.07 (s, 2H), 2.06 - 1.89 (m, 4H), 0.77 (t, J = 7.4 Hz, 6H).13C NMR (126 MHz, CDCI3) 6 178.93, 157.62, 136.94, 135.50, 128.62, 128.34, 128.04, 127.52, 114.71 , 70.04, 53.92, 27.08, 8.32. HRMS (ESI) calculated for C19H23NO2 [M+Na]+: 320.1626, found 320.1621.2-(4-(Benzyloxy)phenyl)-2-ethylbutanethioamide (11)

[0240] To a solution of 2-(4-(Benzyloxy)phenyl)-2-ethylbutanamide (500 mg, 1.7 mmol, 1 equiv.) in anhydrous THF (4 mL, 0.4 M), added Lawesson’s reagent (414 mg, 1 mmol, 1.1 equiv.) at room temperature. Heated to 45 °C for 3 hours. Cooled to room temperature, dissolved in ethyl acetate (10 mL), and washed with saturated sodium bicarbonate (10 mL). Dried the organic layer with anhydrous sodium sulfate. Concentrated under reduced pressure and purified via FCC (gradient of 15% to 50% ethyl acetate in hexanes) to give 2-(4-(Benzyloxy)phenyl)-2- ethylbutanethioamide as a white solid (113 mg, 26% yield). Rf = 0.37 (30% ethyl acetate inhexanes). IR (thin film) v 3395, 3270, 3153, 2971 , 2935, 2875, 1619, 1506, 1454 crrr1.1H NMR (500 MHz, CDCI3) 6 7.56 (s, 1 H), 7.50 - 7.35 (m, 5H), 7.28 - 7.25 (m, 2H), 7.06 - 6.91 (m, 2H), 6.54 (s, 1 H), 5.08 (s, 2H), 2.22 (dq, J = 14.5, 7.3 Hz, 2H), 2.12 - 2.02 (m, 2H), 0.78 (t, J = 7.4 Hz, 6H).13C NMR (126 MHz, CDCI3) 6 216.59, 157.77, 136.86, 135.45, 128.64, 128.61 , 128.08, 127.53, 114.85, 70.05, 58.04, 30.41 , 8.70. HRMS (ESI) calculated for C19H23NOS [M+Na]+: 336.1398, found 336.1393.3-Bromo-6-((tert-butyldimethylsilyl)oxy)-7,7-dimethyloctan-2-one (17a)

[0241] To a mixture of 6-((tert-Butyldimethylsilyl)oxy)-7,7-dimethyloctan-2-one (690 mg, 2.4 mmol, 1 equiv.) and NBS (448 mg, 2.52 mmol, 1.05 equiv.) in CCI4 (3 mL) was added NH4OAC (19 mg, 0.024, 0.1 equiv.) at room temperature. After stirring at 80 °C 86 for 15 minutes, the mixture was cooled to room temperature and dissolved in DCM (10 mL) and water (10 mL). Extracted the aqueous phase with DCM (10 mL, 3x). Dried the combined organic layers over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified vis FCC (1% ether in hexanes) to give 3-Bromo-6-((tert-butyldimethylsilyl)oxy)-7,7-dimethyloctan- 2-one and 1-Bromo-6-((tert-butyldimethylsilyl)oxy)-7,7-dimethyloctan-2-one as a yellow oil (4:1 rr, 559 mg, 64% yield). Rf = 0.35 (2% diethyl ether in hexanes). The mixture of of a-bromoketones (100 mg, 0.27 mmol, 1 equiv.) was added to a solution of 4-chlorothiophenol (8 mg, 0.067 mmol, 0.25 equiv.) and potassium carbonate (10 mg, 0.067 mmol, 0.25 equiv.) in ethanol (0.6 mL) at room temperature. Stirred for 2 hours, then filtered through a thin pad of celite and washed with ethanol (5 mL). Concentrated under reduced pressure and purified by FCC (1% diethyl ether in hexanes) to give 3-Bromo-6-((tert-butyldimethylsilyl)oxy)-7,7-dimethyloctan-2-one as a yellow oil (20:3 rr, 68 mg, 68% yield). IR (thin film) v 2954, 2856, 1720, 1472, 1359 cm’1.1H NMR (500 MHz, CDCI3) 6 4.24 - 4.15 (m, 1 H) 3.32 - 3.23 (m, 1 H), 2.38 (d, J = 3.5 Hz, 3H), 2.29 - 1.71 (m, 4H), 0.98 - 0.92 (m, 9H), 0.92 - 0.82 (m, 9H), 0.13 - 0.02 (m, 6H).13C NMR (126 MHz, CDCI3) 6 201.90, 79.85, 79.78, 55.06, 54.86, 36.01, 35.95, 31.77, 31.48, 31.13, 31.07, 26.48, 26.47, 26.20, 26.15, 26.13, 18.38, -3.28, -3.29, -3.88, -3.90. HRMS (ESI) calculated for Ci6H33O2BrSi [M+Na]+: 387.1331 , found 387.1324.1-(2-(3-(4-(Benzyloxy)phenyl)pentan-3-yl)-5-methylthiazol-4-yl)-4,4-dimethylpentan-3-ol (18)

[0242] To a solution of 2-(4-(Benzyloxy)phenyl)-2-ethylbutanethioamide (256 mg, 0.82 mmol, 1 equiv.) in anhydrous ethanol (1.6 mL, 0.5 M) was added 3-Bromo-6-((tert- butyldimethylsilyl)oxy)-7,7-dimethyloctan-2-one (329 mg, 0.9 mmol, 1.1 equiv.). The reaction washeated at reflux overnight. Solvent was removed under reduced pressure and the crude mixture was dissolved in ethyl acetate (2 mL) and water (2 mL). The aqueous layer was extracted with ethyl acetate (2 mL, 3x). Organic layers were combined and washed with brine (6 mL). Organic layer was dried over anhydrous sodium sulfate. The residue was purified by FCC (10% ethyl acetate in hexanes) to give 1-(2-(3-(4-(Benzyloxy)phenyl)pentan-3-yl)-5-methylthiazol-4-yl)-4,4- dimethylpentan-3-ol as a white solid (257 mg, 67% yield). Rf= 0.14 (20% ethyl acetate in hexanes). IR (thin film) v 3364, 2961 , 2924, 2864, 1607, 1509 cm’1.1H NMR (500 MHz, CDCI3) 5 7.49 - 7.31 (m, 5H), 7.26 - 7.20 (m, 2H), 6.95 - 6.89 (m, 2H), 5.05 (s, 2H), 3.18 (dd, J = 10.6, 1.8 Hz, 1 H), 2.95 (ddd, J = 14.5, 9.4, 4.7 Hz, 1 H), 2.73 (ddd, 87 J = 15.0, 9.1 , 7.3 Hz, 1 H), 2.35 (s, 3H), 2.31 - 2.17 (m, 4H), 1.75 (dddd, J = 13.7, 9.3, 7.4, 1.8 Hz, 1 H), 1.55 (dddd, J = 13.8, 10.6, 9.1 , 4.7 Hz, 1 H), 1.48 - 1.37 (m, 1 H), 0.89 (s, 9H), 0.75 (t, 6H).13C NMR (126 MHz, CDCI3) 5 175.30, 157.14, 146.20, 138.77, 137.15, 131.26, 128.57, 128.41 , 127.94, 127.56, 114.13, 79.02, 77.29, 77.04, 76.79, 69.97, 51.00, 34.92, 33.36, 29.71 , 29.69, 25.62, 23.88, 15.10, 8.30. HRMS (ESI) calculated for C29H39NO2S [M+H]+: 466.2780, found 466.2768.4-(3-(4-(3-Hydroxy-4,4-dimethylpentyl)-5-methylthiazol-2-yl)pentan-3-yl)phenol (19)

[0243] Pd / C (12 mg, 10 wt%) was added in one portion to 1-(2-(3-(4- (Benzyloxy)phenyl)pentan-3-yl)-5-methylthiazol-4-yl)-4,4-dimethylpentan-3-ol (264 mg, 0.57 mmol, 1 equiv.) in anhydrous methanol (6.4 mL, 0.09 M) at room temperature. Filled the reaction vessel with hydrogen gas. Stirred at room temperature overnight. Monitored by TLC. Filtered over a thin pad of celite and washed with methanol (10 mL). Concentrated under reduced pressure and purified via FCC (30% ethyl acetate in hexanes) to give 4-(3-(4-(3-Hydroxy-4,4- dimethylpentyl)-5-methylthiazol-2-yl)pentan-3-yl)phenol as a pale-yellow solid (169 mg, 79% yield). Rf = 0.25 (30% ethyl acetate in hexanes) IR (thin film) v 3367, 2961 , 2925, 2864, 1509, 1455 cm’1.1H NMR (500 MHz, CDCI3) 6 7.71 (s, 1 H), 6.96 (t, J = 7.7 Hz, 2H), 6.44 (d, J = 9.9 Hz, 2H), 3.22 (dd, J = 10.5, 5.3 Hz, 1 H), 2.99 (ddd, J = 14.5, 9.4, 4.7 Hz, 1 H), 2.77 (dt, J = 15.5, 8.2 Hz, 1 H), 2.38 - 2.23 (m, 5H), 2.15 (dq, J = 14.3, 7.3 Hz, 2H), 1.81 (dt, J = 16.3, 8.2 Hz, 1 H), 1.69 - 1.59 (m, 1 H), 1.42 (dd, J = 7.4, 3.5 Hz, 1 H), 0.92 (S, 9H), 0.71 (t, J = 7.3 Hz, 6H).13C NMR (126 MHz, CDCI3) 6 177.50, 155.23, 146.97, 135.82, 130.31 , 127.82, 115.08, 79.06, 77.28, 77.03, 76.78, 50.47, 34.96, 33.34, 28.73, 25.64, 23.74, 14.39, 8.09. HRMS (ESI) calculated for C22H33NO2S [M+H]+: 376.2305, found 376. 2303.Ethyl 4-(4-(3-(4-(3-hydroxy-4,4-dimethylpentyl)-5-methylthiazol-2-yl)pentan-3- yl)phenoxy)butanoate (20a)

[0244] Ethyl 4-bromobutyrate (16 pL, 0.1 mmol, 1.25 equiv.) was added in one portion to a vigorously stirred slurry of 4-(3-(4-(3-Hydroxy-4,4-dimethylpentyl)-5-methylthiazol-2-yl)pentan-3- yl)phenol (31 mg, 0.08 mmol, 1 equiv.) and solid K2CO3 (22 mg, 0.16 mmol, 2 equiv.) in MeCN (0.4 mL) under air. The reaction mixture was heated at reflux overnight, at which point TLC analysis indicated complete consumption of the starting material. The reaction mixture was allowed to cool to room temperature and filtered over a pad of celite via vacuum filtration. The filtrate was collected as a clear solution and concentrated to a clear oil. The residue was purified by FCC (30% ethyl acetate in hexanes) to provide Ethyl 4-(4-(3-(4-(3-hydroxy-4,4-dimethylpentyl)- 5-methylthiazol-2-yl)pentan-3- yl)phenoxy)butanoate as a clear oil (39 mg, 98% yield). Rf= 0.40 89 (30% ethyl acetate in hexanes). IR (thin film) v 3516, 2963, 2872, 1733, 1609, 1510 cm- 1.1H NMR (500 MHz, CDCI3) 5 7.26 - 7.15 (m, 2H), 6.85 - 6.77 (m, 2H), 4.15 (q, J = 7.1 Hz, 2H), 3.99 (t, J = 6.1 Hz, 2H), 3.17 (dd, J = 10.6, 1.8 Hz, 1 H), 2.94 (ddd, J = 14.4, 9.4, 4.7 Hz, 1 H), 2.72 (ddd, J = 15.0, 9.1 , 7.4 Hz, 1 H), 2.51 (t, J = 7.3 Hz, 2H), 2.34 (s, 3H), 2.30 - 2.17 (m, 4H), 2.17 - 2.09 (m, 2H), 1.73 (dddd, J = 13.9, 9.3, 7.4, 1.8 Hz, 1 H), 1.60 - 1.50 (m, 1 H), 1.27 (td, J = 7.2, 3.2 Hz, 4H), 0.88 (s, 9H), 0.73 (td, J = 7.3, 1.0 Hz, 6H).13C NMR (126 MHz, CDCI3) 6 175.33, 173.27, 157.09, 146.14, 138.51 , 131.26, 128.36, 113.77, 78.99, 77.30, 77.05, 76.79, 66.55, 60.39, 50.96, 34.90, 33.35, 30.84, 29.68, 29.66, 25.61 , 24.70, 23.87, 15.08, 14.20, 8.27, 8.26. HRMS (ESI) calculated for C28H43NO4S [M+H]+: 490.2985, found 490.2981.Ethyl 5-(4-(3-(4-(3-hydroxy-4,4-dimethylpentyl)-5-methylthiazol-2-yl)pentan-3- yl)phenoxy)pentanoate (20b)

[0245] Ethyl 5-bromovalerate (16 pL, 0.1 mmol, 1.25 equiv.) was added in one portion to a vigorously stirred slurry of 4-(3-(4-(3-Hydroxy-4,4-dimethylpentyl)-5-methylthiazol-2-yl)pentan-3- yl)phenol (29.2 mg, 0.07 mmol, 1 equiv.) and solid K2CO3 (22 mg, 0.16 mmol, 2 equiv.) in MeCN (0.4 mL) under air. The reaction mixture was heated at reflux overnight, at which point TLC analysis indicated complete consumption of the starting material. The reaction mixture was allowed to cool to room temperature and filtered over a pad of celite via vacuum filtration. The filtrate was collected as a clear solution and concentrated to a clear oil. The residue was purified by FCC (30% ethyl acetate in hexanes) to provide Ethyl 5-(4-(3-(4-(3-hydroxy-4,4-dimethylpentyl)- 5-methylthiazol-2-yl)pentan-3- yl)phenoxy)pentanoate as a clear oil (37 mg, 97% yield,). Rf = 0.35 (30% EtOAc in hexanes). IR (thin film) v 3517, 2963, 2872, 1732, 1609, 1510, 1468 cm- 1 . 1 H NMR (500 MHz, CDCI3) 5 7.23 - 7.14 (m, 2H), 6.85 - 6.75 (m, 2H), 4.14 (q, J = 7.2 Hz, 2H), 4.02 - 3.93 (m, 2H), 3.17 (dd, J = 10.6, 1.8 Hz, 1 H), 2.94 (ddd, J = 14.5, 9.4, 4.7 Hz, 1 H), 2.72 (ddd, J = 14.9, 9.1 , 7.4 Hz, 1 H), 2.43 - 2.37 (m, 2H), 2.34 (s, 3H), 2.28 - 2.16 (m, 4H), 1.87 - 1.78 (m,4H), 1.74 (dddd, J = 13.8, 9.4, 7.4, 1.8 Hz, 1 H), 1.54 (dddd, J = 13.8, 10.6, 9.1 , 4.7 Hz, 1 H), 1.27 (m, 4H), 0.88 (s, 9H), 0.73 (td, J = 7.3, 1.0 Hz, 6H). 13C NMR (126 MHz, CDCI3) 5 175.40, 173.49, 157.21 , 146.13, 138.39, 131.25, 128.35, 113.75, 79.00, 67.20, 60.30, 50.97, 34.91 , 33.98, 33.35, 29.69, 29.67, 28.74, 25.61 , 90 23.87, 21.70, 15.08, 14.25, 8.28, 8.27. HRMS (ESI) calculated for C29H45NO4S [M+H]+ : 504.3142, found 504.3134.Ethyl 6-(4-(3-(4-(3-hydroxy-4,4-dimethylpentyl)-5-methylthiazol-2-yl)pentan-3- yl)phenoxy)hexanoate (20c)

[0246] Ethyl 6-bromohexanoate (19 pL, 0.1 mmol, 1.25 equiv.) was added in one portion to a vigorously stirred slurry of 4-(3-(4-(3-Hydroxy-4,4-dimethylpentyl)-5-methylthiazol-2-yl)pentan-3- yl)phenol (28 mg, 0.07 mmol, 1 equiv.) and solid K2CO3 (22 mg, 0.16 mmol, 2 equiv.) in MeCN (0.4 mL) under air. The reaction mixture was heated at reflux overnight, at which point TLC analysis indicated complete consumption of the starting material. The reaction mixture was allowed to cool to room temperature and filtered over a pad of celite via vacuum filtration. The filtrate was collected as a clear solution and concentrated to a clear oil. The residue was purified by FCC (30% ethyl acetate in hexanes) to provide Ethyl 6-(4-(3-(4-(3-hydroxy-4,4-dimethylpentyl)- 5-methylthiazol-2-yl)pentan-3- yl) phenoxy) hexanoate as a clear oil (38 mg, 99% yield). Rf = 0.42 (30% ethyl acetate in hexanes). IR (thin film) v 3488, 2939, 2869, 1731 , 1609, 1510, 1467 cm- 1 . 1 H NMR (500 MHz, CDCI3) 5 7.23 - 7.15 (m, 2H), 6.85 - 6.77 (m, 2H), 4.23 - 4.01 (m, 2H), 3.94 (t, J = 6.4 Hz, 2H), 3.17 (dd, J = 10.6, 1.8 Hz, 1 H), 2.93 (ddd, J = 14.5, 9.4, 4.7 Hz, 1 H), 2.72 (ddd, J = 14.9, 9.1 , 7.4 Hz, 1 H), 2.33 (d, J = 7.4 Hz, 5H), 2.28 - 2.11 (m, 4H), 1.93 - 1.62 (m, 5H), 1.59 - 1.44 (m, 3H), 1.27 (td, J = 7.2, 3.6 Hz, 4H), 0.87 (s, 9H), 0.73 (t, J = 7.9 Hz, 6H). 13C NMR (126 MHz, CDCI3) 5 175.39, 173.66, 157.29, 146.12, 138.32, 131.25, 128.33, 113.75, 78.98, 67.46, 60.23, 50.95, 34.90, 34.27, 33.35, 29.69, 29.02, 25.70, 25.61 , 24.73, 23.87, 15.08, 14.25, 8.28. HRMS (ESI) calculated for C30H47NO4S [M]+ : 518.3298, found 518.3297.N-Hydroxy-4-(4-(3-(4-(3-hydroxy-4,4-dimethylpentyl)-5-methylthiazol-2-yl)pentan-3- yl)phenoxy)butanamide (7a; AC-340)

[0247] Hydroxylamine (1.67 mL, 25 mmol, aq. 50%wt solution, 500 equiv.), and 3 M KOH (aq.) (0.12 mL, 0.35 mmol, 7 equiv.) were added in that order to a vigorously stirring solution of Ethyl 4-(4-(3-(4-(3-hydroxy-4,4-dimethylpentyl)-5-methylthiazol-2-yl)pentan-3- yl)phenoxy)butanoate (32.8 mg, 0.067 mmol, 1 equiv.) at OoC in a 1 :1 MeOH / THF mixture (6.5 mL) under air. The reaction was left to warm slowly to room temperature overnight. After 24 hours,volatiles were removed in vacuo and the residue was and dissolved in HPLC grade ethyl acetate. Washed organic layer with a sodium phosphate buffer (pH=7) (5 mL). Concentrated in vacuo and the residue was purified by reversed-phase chromatography on octadecyl-functionalized silica gel using a gradient of 60-95% MeOH:H2O over a period of 20 minutes as eluent to provide AC-340 as a fine white powder (13.7 mg) in 43% yield after lyophilization. IR (thin film) v 3213, 2963, 2873, 1652, 1510, 1469 cm ’1.1H NMR (500 MHz, DMSO-cfe) 6 10.39 (s, 1 H), 8.68 (s, 1 H), 7.17 - 7.11 (m, 2H), 6.86 - 6.79 (m, 2H), 4.42 (d, J = 6.4 Hz, 1 H), 3.91 (t, J = 6.3 Hz, 2H), 2.93 (ddd, J = 10.5, 6.4, 1.7 Hz, 1 H), 2.84 (ddd, J = 14.4, 9.5, 4.4 Hz, 1 H), 2.62 (ddd, J = 15.0, 9.2, 7.3 Hz, 1 H), 2.21 (s, 3H), 2.12 (p, J = 7.4 Hz, 6H), 1.90 (p, J = 6.7 Hz, 2H), 1.65 - 1.55 (m, 1 H), 1.42 - 1.28 (m, 1 H), 0.77 (s, 9H), 0.64 (td, J = 7.3, 1.5 Hz, 6H).13C NMR (126 MHz, DMSO- cfe) 6 174.43, 169.07, 157.20, 146.03, 138.25, 132.04, 128.49, 114.31 , 77.18, 67.11 , 50.84, 35.14, 33.82, 29.47, 29.22, 26.33, 25.33, 23.78, 15.28, 8.66. HRMS (ESI-) calculated for C26H40N2O4S [M-H]’ : 475.2636, found 475.2637. Purity >99% by LC-MS, tr = 11.5 minutes.N-Hydroxy-5-(4-(3-(4-(3-hydroxy-4,4-dimethylpentyl)-5-methylthiazol-2-yl)pentan-3- yl)phenoxy)pentanamide (7b; AC-339)

[0248] Hydroxylamine (1.67 mL, 25 mmol, aq. 50%wt solution, 500 equiv.), and 3 M KOH (aq.) (0.12 mL, 0.35 mmol, 7 equiv.) were added in that order to a vigorously stirring solution of Ethyl 5-(4-(3-(4-(3-hydroxy-4,4-dimethylpentyl)-5-methylthiazol-2-yl)pentan-3- yl)phenoxy)pentanoate (30 mg, 0.059 mmol, 1 equiv.) at 0°C in a 1 :1 MeOH / THF mixture (6.5 mL) under air. The reaction was left to warm slowly to room temperature overnight. After 24 hours, volatiles were removed in vacuo and the residue was purified by reversed-phase chromatography on octadecyl-functionalized silica gel using a gradient of 50-95% MeOH:H2O over a period of 20 minutes as eluent to provide N-Hydroxy-5-(4-(3-(4-(3-hydroxy-4,4-dimethylpentyl)-5- methylthiazol-2-yl)pentan-3- yl)phenoxy)pentanamide (AC-339) as a fine white powder (27 mg) in 92% yield after lyophilization. IR (thin film) v 3231 , 2939, 2873, 1643, 1510, 1393 cm- 1 . 1 H NMR (500 MHz, DMSO) 5 7.19 - 7.09 (m, 2H), 7.02 - 6.70 (m, 2H), 4.44 (s, 1 H), 3.92 (t, J = 6.2 Hz, 2H), 2.95 (d, J = 10.4 Hz, 1 H), 2.85 (ddd, J = 14.4, 9.4, 4.5 Hz, 1 H), 2.71 - 2.57 (m, 1 H), 2.23 (s, 3H), 2.14 (q, J = 7.3 Hz, 4H), 1.97 (s, 2H), 1.73 - 1.54 (m, 5H), 1.44 - 1.31 (m, 1 H), 0.78 (s, 9H), 0.65 (, J = 7.3 Hz, 6H). 13C NMR (126 MHz, DMSO) 5 174.45, 168.88, 157.30, 146.02, 138.12, 132.03, 128.46, 128.43, 114.27, 77.18, 67.40, 50.83, 35.14, 33.81 , 32.54, 29.46, 28.74, 26.33, 23.78, 22.39, 15.27, 8.67. HRMS (ESI+) calculated for C27H42N2O4S [M+H]+ : 491.2938, found 491.2938. Purity >96% by LC-MS, tr = 11.84 minutes.N-Hydroxy-6-(4-(3-(4-(3-hydroxy-4,4-dimethylpentyl)-5-methylthiazol-2-yl)pentan-3- yl)phenoxy)hexanamide (7c; AC-341)

[0249] Hydroxylamine (1.67 mL, 25 mmol, aq. 50%wt solution, 500 equiv.), and 3 M KOH (aq.) (0.12 mL, 0.35 mmol, 7 equiv.) were added in that order to a vigorously stirring solution of Ethyl 6-(4-(3-(4-(3-hydroxy-4,4-dimethylpentyl)-5-methylthiazol-2-yl)pentan-3- yl)phenoxy)hexanoate (29.8 mg, 0.057mmol, 1 equiv.) at OoC in a 1 :1 MeOH / THF mixture (6.5 mL) under air. The reaction was left to warm slowly to room temperature overnight. After 24 hours, volatiles were removed in vacuo and the residue was purified by reversed-phase chromatography on octadecyl-functionalized silica gel using a gradient of 60-95% MeOH:H2O over a period of 20 minutes as eluent to provide N-Hydroxy-6-(4-(3-(4-(3-hydroxy-4,4-dimethylpentyl)-5- methylthiazol-2-yl)pentan-3- yl)phenoxy)hexanamide ( AC-341) (23.1 mg, 80% yield) as a fine white powder after lyophilization. I R (thin film) v 3213, 2937, 2865, 1633, 1509 cm- 1 . 1 H NMR (500 MHz, DMSO) 5 7.25 - 7.04 (m, 2H), 6.89 - 6.68 (m, 2H), 3.89 (t, J = 6.5 Hz, 2H), 3.00 - 2.88 (m, 1 H), 2.84 (ddd, J = 14.4, 9.7, 4.5 Hz, 1 H), 2.62 (dt, J = 15.6, 8.5 Hz, 1 H), 2.21 (s, 3H), 2.12 (q, J = 7.3 Hz, 4H), 1.90 (s, 1 H), 1.66 (p, J = 6.7 Hz, 2H), 1.58 (dd, J = 14.7, 7.1 Hz, 1 H), 1.50 (q, J = 7.9 Hz, 2H), 1.35 (qt, J = 9.6, 4.1 Hz, 3H), 0.77 (s, 9H), 0.72 - 0.58 (m, 6H).13C NMR (126 MHz, DMSO) 5 174.46, 168.99, 157.33, 146.02, 138.10, 132.04, 128.47, 114.26, 77.17, 93 67.67, 50.83, 35.14, 33.81 , 32.94, 29.46, 29.00, 26.33, 25.75, 25.35, 23.78, 15.27, 8.66. HRMS (ESI- ) calculated for C28H43N2O4S [M-H]- : 503.2949, found 503.2948. Purity >98% by LC-MS, tr = 12.40 minutes.

Claims

WHAT IS CLAIMED IS:

1. A compound of formula I or a pharmaceutically acceptable salt or solvate thereofwherein:W is a 5 or 6 membered heteroaryl group comprising at least one nitrogen atom as a heteroatom in the heteroaryl group, and the heteroaryl group has a methyl substitution, when possible, adjacent to Y;Xi is CO or O,X2 is NH or CH2, wherein when Xi is CO, X2 is NH and when Xi is O, X2 is CH2;Y is O or CH2; n is an integer of from 2 to 5; and when Xi is CO, W is a 6 membered heteroaryl group with only one nitrogen atom or is a 5 membered heteroaryl group. wherein when Xi is O, W is a 5 membered heteroaryl group and when Xi is CO, W is a 6 membered heteroaryl group with only one nitrogen atom or is a 5 membered heteroaryl group2. The compound of claim 1 , wherein n is an integer of from 2 to 4 when Xi is O and n is an integer of from 3 to 5 when Xi is CO.

3. The compound of claim 1 or 2, wherein W is selected from the group consisting of4. The compound of any one of claims 1 to 3, wherein5. The compound of claim 4, whereinwhen Xi is CO and W iswhen Xi is O.

6. The compound of any one of claims 1 to 5, wherein the compound isThe compound of any one of claims 1 to 6, for use in the treatment of a cancer selected from the group consisting of leukemia, non-small cell lung cancer, colon cancer, central nervous system cancer, melanoma, ovaria cancer, renal cancer, prostate cancer, and breast cancer.

8. The compound of any one of claims 1 to 6, for use in the manufacture of a medicament for treating a cancer selected from the group consisting of leukemia, non-small cell lung cancer, colon cancer, central nervous system cancer, melanoma, ovaria cancer, renal cancer, prostate cancer, and breast cancer.

9. The compound of any one of claims 1 to 6, for use in the treatment of psoriasis.

10. The compound of any one of claims 1 to 6, for use in the manufacture of a medicament for the treatment of psoriasis.

11. A pharmaceutical composition comprising the compound as defined in any one of claims 1 to 6 and a pharmaceutically acceptable excipient.

12. The pharmaceutical composition of claim 11 , for use in the treatment of a cancer selected from the group consisting of leukemia, non-small cell lung cancer, colon cancer, central nervous system cancer, melanoma, ovaria cancer, renal cancer, prostate cancer, and breast cancer.

13. The pharmaceutical composition of claim 11 , for use in the manufacture of a medicament for treating a cancer selected from the group consisting of leukemia, non-small cell lung cancer, colon cancer, central nervous system cancer, melanoma, ovaria cancer, renal cancer, prostate cancer, and breast cancer.

14. The pharmaceutical composition of claim 11 , for use in the treatment of psoriasis.

15. The pharmaceutical composition of claim 11 , for use in the manufacture of a medicament for the treatment of psoriasis.

16. Use of the compound of any one of claims 1 to 6 or of the pharmaceutical composition of claim 11 , in the treatment of a cancer selected from the group consisting of leukemia, non- small cell lung cancer, colon cancer, central nervous system cancer, melanoma, ovaria cancer, renal cancer, prostate cancer, and breast cancer.

17. Use of the compound of any one of claims 1 to 6 or of the pharmaceutical composition of claim 11 , in the manufacture of a medicament for the treatment of a cancer selected from the group consisting of leukemia, non-small cell lung cancer, colon cancer, central nervoussystem cancer, melanoma, ovaria cancer, renal cancer, prostate cancer, and breast cancer.

18. Use of the compound of any one of claims 1 to 6 or of the pharmaceutical composition of claim 11 , in the treatment of psoriasis.

19. Use of the compound of any one of claims 1 to 6 or of the pharmaceutical composition of claim 11 , in the manufacture of psoriasis.

20. A method of treating a patient with a cancer selected from the group consisting of leukemia, non-small cell lung cancer, colon cancer, central nervous system cancer, melanoma, ovaria cancer, renal cancer, prostate cancer, and breast cancer, comprising administering to the patient the compound as defined in any one of claims 1 to 6 or the pharmaceutical composition as defined in claim 11.

21. A method of treating a patient with psoriasis, comprising administering to the patient the compound as defined in any one of claims 1 to 6 or the pharmaceutical composition as defined in claim 11.

22. A method of treating a patient with a cancer selected from the group consisting of leukemia, non-small cell lung cancer, colon cancer, central nervous system cancer, melanoma, ovaria cancer, renal cancer, prostate cancer, and breast cancer, by administering to the patient the compound as defined in any one of claims 1 to 6 or the pharmaceutical composition as defined in claim 11 , and by also administering to the patient an immune checkpoint blockade agent.

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