Targeting HIF2α in kidney cancer

Abstract

The instant discloses provides compositions of matter and methods of using same and is directed to antagonists of Hypoxia-inducible factor 2α (HIF2α) transcription factor. HIF2α is involved in the adaptation of cancer cells, including clear cell renal cell carcinoma (ccRCC), to low oxygen conditions (hypoxia) and plays a role in promoting tumor growth and angiogenesis. Therefore, pharmacologic inhibition of HIF2α holds a novel therapeutic strategy for cancers driven by HIF2α signaling. Embodiments disclosed herein provide novel HIF2α antagonists having distinctive inhibitory mechanisms compared to previously reported HIF2α antagonists. The claimed compounds provide promising alternatives for addressing drug resistance and present a unique approach in inhibiting HIF2α-related processes.

Description

TARGETING HIF2a IN KIDNEY CANCERCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of the filing date of U.S. Provisional Application No. 63 / 546,096, filed October 27, 2023. The content of this this earlier filed application is hereby incorporated by reference herein in its entirety.BACKGROUND OF THE INVENTIONField of the Invention.

[0002] Hypoxia-Inducible Factor 2 alpha (HIF2a) is a transcription factor that plays an essential role in mediating cellular responses to hypoxia. HIF2a structure and function have attracted significant interest, as misregulation of the physiological and pathological processes influenced by HIF2o are implicated in the development and progression of several forms of cancer, including renal cancer. HIF2a belongs to the basic helix-loop-helix (bHLH) Per-Arnt-Sim (PAS) family of transcription factors. The protein consists of several functional domains, including the N-terminal bHLH domain that enables DNA binding, two Per-Arnt-Sim (PAS) domains (PAS-A and PAS-B) involved in heterodimerization with its partner Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT), and a C-terminal transactivation domain that facilitates transcriptional activity. Notably, the PAS-B domain has been identified as a regulatory site susceptible to ligand interactions, offering a potential target for therapeutic interventions.

[0003] Under normoxic conditions, HIF2a is hydroxylated by prolyl hydroxylase enzymes, marking it for proteasomal degradation. However, during hypoxia, these hydroxylations are impaired, allowing HIF2a to evade degradation. Stabilized HIF2a forms a heterodimer with ARNT and translocates to the nucleus, where it binds to hypoxia-responsive elements (HREs) in the promoters of genes involved in angiogenesis, erythropoiesis, glucose metabolism, and cell survival. Tumor cells depend on these genes to ensure their growth and survival in the hostile tumor microenvironment. HIF2a also contributes to the epithelial-mesenchymal transition (EMT), a process facilitating metastasis by enhancing cell mobility and invasiveness.

[0004] Clear cell renal cell carcinoma (ccRCC) serves as a great example of HIF2a playing a role in cancer progression. Mutations in the tumor suppressor Von Hippel- Lindau (VHL) gene are prevalent in ccRCC. Resultant mutant VHL protein is unable to exert the regulatory function of targeting of HIF2a for degradation, resulting in the accumulation of HIF2o. This leads to the aberrant expression of HIF2a target genes, fueling tumor development. A number of pharmaceutical agents have been developed targeting HIF2a to treat cancer. For example, Belzutifan (MK-6482 also known as WELIREG, Merck), is a U.S. Food and Drug Administration (FDA) approved HIF2a inhibitor. It disrupts HIF2a activity by binding to its PAS-B domain, inhibiting downstream gene expression crucial for tumor growth. Belzutifan is intended for adult patients with VHL disease who need treatment for associated conditions like renal cell carcinoma (RCC), central nervous system (CNS) hemangioblastomas, or pancreatic neuroendocrine tumors (pNET) that do not necessitate immediate surgery and has recently be shown to be superior to mTOR inhibition in patients with metastatic ccRCC who failed immune checkpoint and anti-angiogenic therapies. Therefore, HIF2a inhibition through pharmacologic small molecules offers an attractive therapeutic approach for HIF2a-driven cancers.BRIEF SUMMARY OF THE INVENTION

[0005] Hypoxia-Inducible Factor 2a (HIF2a) transcription factor is involved in the adaptation of cancer cells, including clear cell renal cell carcinoma (ccRCC), to low oxygen conditions (hypoxia) and plays a role in promoting tumor growth and angiogenesis.

[0006] Embodiments disclosed herein are directed to in silico screening using the PAS-B domain of HIF2a. As a result, we have identified compounds, such as Compound-c2, that are novel antagonist of HIF2a and demonstrated that it specifically binds to and disrupts HIF2a interaction with the molecular chaperone Heat shock protein-70 (Hsp70), which consequently leads to the proteasomal degradation of HIF2a and induces apoptosis in ccRCC. According to embodiments disclosed herein are methods for inhibiting Hypoxia-Inducible Factor 2 alpha (HIF2a) in a patient (or mammal) in need thereof. The methods comprise administering a therapeutically effective amount of at least one Hypoxia-Inducible Factor 2 alpha (HIF2a) antagonistcompound and the novel antagonists of HIF2a have the following structural formula (I):wherein X is a halogen, an alkyl, a vinyl, an alkynyl or an aryl; Y is a halogen, an alkyl, a vinyl, an alkynyl or an aryl; R1is an alkyl or an aryl; and R2is an alkyl or an aryl, or a pharmaceutically acceptable salt thereof.

[0007] According to embodiments disclosed herein are methods for inhibiting Hypoxia-Inducible Factor 2 alpha (HIF2a) in a patient in need thereof. The methods comprise administering a therapeutically effective amount of at least one the followingHypoxia-Inducible Factor 2 alpha (HIF2a) antagonist compounds:or a pharmaceutically acceptable salt thereof.

[0008] According to embodiments disclosed herein, the methods for inhibiting Hypoxia-Inducible Factor 2 alpha (HIF2a) in a patient in need thereof include the administration of a therapeutically effective amount of at least one of the following compounds: 3-ethyl-4-(p-fluorobenzoylimino)-3,4-dihydro-2(1 / 7)-quinazolinone, 4- bromo-N-[3-ethyl-2-oxo-2,3 dihydro4(1 H)quinazolinyliden]benzenecarboxamide), and tert-butyl N-[3-(4-{[(4E)-3-ethyl-2-oxo-1 ,2,3,4-tetrahydroquinazolin-4-ylidene]carbamoyl} phenyl)prop-2-yn-1-yl]carbamate), or a pharmaceutically acceptable salt thereof.

[0009] According to embodiments disclosed herein, the methods for inhibiting Hypoxia-Inducible Factor 2 alpha (HIF2o) in a patient in need thereof include one more additional therapies. According to embodiments disclosed herein, the methods for inhibiting Hypoxia-Inducible Factor 2 alpha (HIF2a) in a patient in need thereof include therapies comprise chemotherapy, cancer immunotherapy, surgical removal of all or part of the cancerous tissue, or radiation therapy.

[0010] According to embodiments disclosed herein are methods for inhibiting Hypoxia-Inducible Factor 2 alpha (HIF2a) in a patient in need thereof. The methods comprise administering a therapeutically effective amount of at least one Hypoxia- Inducible Factor 2 alpha (HIF2a) antagonist compound the novel antagonists of HIF2a have the following structural formula:or a pharmaceutically acceptable salt thereof.

[0011] According to embodiments disclosed herein, the methods for inhibiting Hypoxia-Inducible Factor 2 alpha (HIF2a) in a patient in need thereof include the administration of a therapeutically effective amount of at least one of the following compounds: 2-aminobenzonitrile, 3,4-dihydro-4-imino-3-ethylquinazolin-2(1 H)-one, and 2-(3-ethylureido)benzonitrile, or a pharmaceutically acceptable salt thereof.

[0012] According to embodiments disclosed herein are new Hypoxia-Inducible Factor 2 alpha (HIF2a) antagonist compound comprising the structural formula I:wherein X is a halogen, an alkyl, a vinyl, an alkynyl or an aryl; Y is a halogen, an alkyl, a vinyl, an alkynyl or an aryl; R1is an alkyl or an aryl; and R2is an alkyl or an aryl, or a pharmaceutically acceptable salt thereof. According to embodiments disclosed herein, the new HIF2a antagonist compound include 3-ethyl-4-(p-fluorobenzoylimino)- 3,4-dihydro-2(1 / - / )-quinazolinone,4-bromo-N-[3-ethyl-2-oxo-2,3 dihydro4(1 H)quinazolinyliden]benzenecarboxamide), and tert-butyl N-[3-(4-{[(4E)-3- ethyl-2-oxo-1 ,2,3,4-tetrahydroquinazolin-4-ylidene] carbamoyl} phenyl)prop-2-yn-1- yl]carbamate), or a pharmaceutically acceptable salt thereof. According to other embodiments disclosed herein are new HIF2a antagonist compound that include the following compounds:or a pharmaceutically acceptable salt thereof.

[0013] According to other embodiments disclosed herein are new HIF2a antagonist compound that include 2-aminobenzonitrile, 3,4-dihydro-4-imino-3-ethylquinazolin- 2(1 H)-one, and 2-(3-ethylureido)benzonitrile, or a pharmaceutically acceptable salt thereof.

[0014] According to other embodiments disclosed herein are methods of proteasomal degradation of HIF2a in a patient in need thereof. The methods comprise administering a therapeutically effective amount of at least one of the HIF2a antagonist compounds disclosed herein. According to other embodiments disclosed herein are methods of reducing the interaction of HIF2a and a chaperone Heat shock protein-70 (Hsp70) in a patient in need thereof. The methods comprise administering atherapeutically effective amount of at least one of the HIF2a antagonist compounds disclosed herein.

[0015] According to an embodiment disclosed herein, 3-ethyl-4-(p- fluorobenzoylimino)-3,4-dihydro-2(1H)-quinazolinone (i.e., Compound-c2) was identified as a novel antagonist of HIF2a and demonstrated that it specifically binds to and disrupts HIF2a interaction with the molecular chaperone Heat shock protein-70 (Hsp70). This consequently leads to the proteasomal degradation of HIF2a and induces apoptosis in ccRCC.

[0016] The novel antagonists disclosed herein have the ability to cause degradation of HIF2a and induce apoptosis in renal cancer. The embodiments disclosed herein highlight the distinctive mechanism (i.e., the subject compounds antagonistically disrupt the formation of the HIF2a - Hsp70 protein complex) of the novel antagonists HIF2a compounds, as compared to previously reported HIF2a antagonists. The novel HIF2a antagonists compounds are useful in drug resistance and present a unique approach in inhibiting HIF2a-related processes.

[0017] The transcription factor Hypoxia-Inducible Factor 2a (HIF2a) plays a crucial role in cancer cell adaptation to hypoxic conditions, particularly in clear cell renal cell carcinoma (ccRCC), promoting tumor growth and angiogenesis. Targeting HIF2a through pharmacologic inhibition offers a novel therapeutic strategy for HIF2a-driven cancers. Using an in silico approach, we identified Compound-c2 as a selective inhibitor that binds to the Per-Arnt-Sim-B (PAS-B) domain of HIF2a. Notably, Compound-c2 disrupts the interaction between HIF2a and the molecular chaperone Hsp70, leading to proteasomal degradation of HIF2a and the induction of apoptosis in ccRCC. This distinctive inhibitory mechanism sets Compound-c2 apart from previous HIF2a antagonists, positioning it as a promising alternative with potential applications in addressing drug resistance and providing a unique approach to inhibit HIF2a-related processes.

[0018] Embodiments of the present disclosure, briefly summarized above and discussed in greater detail below, can be understood by reference to the illustrative embodiments of the disclosure depicted in the appended drawings. However, the appended drawings illustrate only typical embodiments of the disclosure and are therefore not to be considered limiting of scope, for the disclosure may admit to other equally effective embodiments.BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0019] Non-limiting and non-exhaustive features will be described with reference to the following figures, wherein like reference numerals refer to like parts throughout the various figures. The figures below were not intended to be drawn to any precise scale with respect to size, angular relationship, or relative position.

[0020] FIG. 1A presents a screening and identification schematic of HIF2a antagonist and intramolecular interfaces 1 , 2, 4, and 5 on ARNT (blue) and HIF2a (green) (PDB:4ZP4). Interface 3 is the intermolecular junction between ARNT and HIF2a.

[0021] FIG. 1 B presents a detailed view of binding pocket 2 on the HIF2a PAS-B domain.

[0022] FIG. 1C presents a detailed view of binding pocket 5 on the HIF2a PAS-A domain.

[0023] FIG. 1 D presents a schematic workflow of in silico screening of a library of drug like compounds (diverse-lib, -100,000 compounds) using Autodock Vina on the MTiOpenScreen server. Potential HIF2a PAS-B and PAS-A inhibitors (c-compounds and i-compounds, respectively) were identified and further analyzed in cell based assays.

[0024] FIG. 2A presents Compound-c2 mediated inhibition and proteasomal degradation of HIF2a via two V7- / L-null ccRCC cell lines (A498, 786-0), VHL-positive ccRCC cell line (Caki-1 ), and normal kidney HEK293 cells were treated with the indicated amounts of Compound-c2 for 24hrs. The effect of Compound-c2 on cell viability was assessed by MTT assay. Error bars represent the standard deviation (S.D.) of three independent experiments. A Student’s t-test was performed to assess statistical significance compared to DMSO vehicle control (*P < 0.05; **P < 0.01 ).

[0025] FIG. 2B presents the treatment of 786-0 cells with the indicated amounts of Compound-c2 for 24hrs. Induction of apoptosis was assessed by immunoblotting. GAPDH was used as a loading control.

[0026] FIG. 2C presents luminescence from 786-0 cells transiently expressing VEGF-luciferase reporter for 24hrs, treated with 30pM of Compound-c2 for an additional 8hrs. siRNA targeting HIF2a was used as a control. Error bars represent the standard deviation (S.D.) of three independent experiments. A Student’s t-test was performed to assess statistical significance (*P < 0.05).

[0027] FIG. 2D presents VEGF mRNA expression that was determined by RT- qPCR in 786-0 cells following treatment with the indicated amounts of Compound-c2 for 24hrs. Bars represent the ratio of Compound-c2VEGF mRNA:cyclophilin mRNA normalized to the OpM control. Error bars represent the standard deviation (S.D.) of three independent experiments. A Student’s t-test was performed to assess statistical significance (**P < 0.01).

[0028] FIG. 2E presents the treatment of 786-0 cells with the indicated amounts of Compound-c2 for 24hrs. The stability of HIF2a was assessed by immunoblotting. GAPDH was used as a loading control.

[0029] FIG. 2F presents the treatment of 786-0 cells with 30pM Compound-c2 for the indicated durations. The stability of HIF2a was assessed by immunoblotting. GAPDH was used as a loading control.

[0030] FIG. 2G presents treatment of OS-RC-2 cells with 30pM Compound-c2 for the indicated length of time. The stability of HIF2a was assessed by immunoblotting. GAPDH was used as a loading control.

[0031] FIG. 2H presents the addition of 50nM Bortezomib to 786-0 cells for 1 hr followed by addition of 30pM Compound-c2 for 4hr. The 786-0 cells were also treated individually with Bortezomib or Compound-c2. The stability of HIF2ct was assessed by immunoblotting. GAPDH was used as a loading control. LE, long exposure. SE, short exposure.

[0032] FIG. 3A is directed to the specificity and selectivity of Compound-c2 for HIF2a wherein Caki-2 cells were treated with 30pM Compound-c2 for the indicated durations. The stability of HIF1a was assessed by immunoblotting. GAPDH was used as a loading control.

[0033] FIG. 3B presents lysate from 786-0 cells was challenged with the indicated amounts of Biotin-Compound-c2 for 1 hr. HIF2a binding, and input was assessed by immunoblotting.

[0034] FIG. 3C presents empty vector (EV) and HA-HIF2a were transiently expressed in HEK293 cells and lysates were challenged with the indicated amounts of Biotin-Compound-c2 for 1hr. HIF2o binding to Compound-c2 was assessed by immunoblotting.

[0035] FIG. 3D presents amino acid sites within the HIF2a PAS-B domain predicted to contact Compound-c2 (PDB:4ZP4).

[0036] FIG. 3E presents HEK293 cells that were transiently transfected with indicated HA-HIF2a mutants. Lysates that were challenged with 1 pM of Biotin- Compound-c2 for 1 hr and HIF2a binding to Compound-c2 was assessed by immunoblotting. Densitometry was performed using Photoshop v.23.5.1 to quantify Western blot band signal intensity. Bars represent the mean signal intensity values of HIF2a input:HIF2a pulldown for three independent measurements. Tukey’s multiple comparisons test was used to determine statistical significance compared to WT (**P < 0.01 ). (See also FIG. S3B-S3C).

[0037] FIG. 4A presents identification of the Compound-c2 pharmacophore chemical structures of the parent Compound-c2 (center circle) and intermediate compounds (outer circle, Compounds-v, w, x, y, and z).

[0038] FIG. 4B presents 786-0 cells that were treated for 24hrs with DMSO (vehicle control) or 10pM of the Compounds-v, w, x, y, and z. Induction of apoptosis was assessed by immunoblotting. GAPDH was used as a loading control.

[0039] FIG. 4C presents A498 cells that were treated for 24hrs with DMSO (vehicle control) or 10pM of the Compounds-y, z, or c2. Induction of apoptosis was assessed by immunoblotting. GAPDH was used as a loading control.

[0040] FIG. 4D presents V7- / L-null ccRCC cell lines (A498, 786-0), VHL- positive ccRCC cell line (Caki-1 ), and normal renal epithelial HK-2 cells were treated with the indicated amounts of Compound-y for 24hrs. The effect of Compound-y on cell viability was assessed by MTT assay. Error bars represent the standard deviation (S.D.) of three independent experiments. A Student’s t-test was performed to assess statistical significance compared to DMSO vehicle control (**P < 0.01 , ***P < 0.001 , ****P < 0.0001).

[0041] FIG. 4E presents A498, 786-0 VHL-null ccRCC cell lines, Caki-1 VHL- positive ccRCC cell line, and normal renal epithelial HK-2 cells were treated with the indicated amounts of Compound-z for 24hrs. The effect of Compound-z on cell viability was assessed by MTT assay. Error bars represent the standard deviation (S.D.) of three independent experiments. A Student’s t-test was performed to assess statistical significance compared to DMSO vehicle control (*P < 0.05; **P < 0.01 , ***P < 0.001 ).

[0042] FIG. 4F presents predicted Compound-z contact sites within the HIF2a PAS-B domain (PDB:4ZP4).

[0043] FIG. 5A presents Compound-c2 disrupts Hsp70 chaperoning of HIF2a, wherein HIF2a was immunoprecipitated (IP) from 786-0 cell lysates using anti-HIF2aantibody (Ab). Interaction with Hsp70 was assessed by immunoblotting. Immunoglobulin G (IgG) was used as a control. GAPDH was used as a loading control.

[0044] FIG. 5B presents HA-HIF2ct was co-expressed with FLAG tagged domains of Hsp70 depicted in the schematic (left) in HEK293 cells: Hsp70 full length (FL), nucleotide binding domain (NTB), and substrate binding domain (SBD), or empty vector (EV) as a control. Binding of Hsp70 domains with HIF2a was assessed by immunoblotting. GAPDH was used as a loading control.

[0045] FIG. 5C presents 786-0 cells that were treated with 10pM of the Hsp70 inhibitor JG98 for the indicated times. The stability of HIF2a was assessed by immunoblotting. Phospho-AKT and AKT client levels were used as a positive control for the Hsp70 inhibitor. GAPDH was used as a loading control.

[0046] FIG. 5D presents 50nM Bortezomib (BZ) that was added to 786-0 cells for 1 hr followed by addition of 10pM JG98 for 4hrs. Additionally, 786-0 cells were treated individually with Bortezomib or JG98. The stability of HIF2a was assessed by immunoblotting. GAPDH was used as a loading control.

[0047] FIG. 5E presents HIF2a that was immunoprecipitated (IP) from untreated and treated 786-0 cells with 30pM Compound-c2 for 2hrs. Anti-HIF2a and immunoglobulin G (IgG) (control) antibodies (Ab) were used to examine HIF2a interaction with Hsp70 by immunoblotting. GAPDH was used as a loading control.

[0048] FIG. 5F presents HIF2a that was immunoprecipitated (IP) from 786-0 cells treated with DMSO (vehicle control) or 10pM of the Compounds-y or z for 24hrs. Interaction of HIF2a with Hsp70 as well as induction of apoptosis was assessed by immunoblotting. GAPDH was used as a loading control.

[0049] FIG. 5G presents HEK293 cells transiently expressing WT HA-HIF2a and S246A, H248A, and F280A mutants were treated with 10|JM of Hsp70 inhibitor, JG98, for the indicated time points. HA-HIF2a protein levels were examine by immunoblotting. GAPDH was used as a loading control.

[0050] FIG. 5H presents a schematic representation of targeting HIF2a by Compounds-c2 and -z. VHL-null kidney tumors exhibit elevated HIF2a levels, which is protected from proteasomal degradation by the molecular chaperone Hsp70 (left). Stable HIF2a translocates to the nucleus to upregulate genes associated with tumor survival. Treatment with Compound-c2 or Compound-z (right) dissociates the HIF2a:Hsp70 complex, causing HIF2a proteasomal degradation and subsequent apoptosis of cancer cells.

[0051] FIG. 6 presents a Primer Table listing the restriction sites that are highlighted in boldface, italics, and underline (lines 33-37); the mutated sequences are highlighted in boldface and underline (lines 5-32); and the epitope sequences are highlighted in boldface and italics (lines 33 and 36).

[0052] FIG. S1A-S1 E are directed to the development and characterization of HIF2a antagonists as related to FIG. 1A-1D: FIG. S1A presents in silico screen identified 23-compounds with potential binding properties to site 2 of HIF2a PAS-B. They are labelled as Compounds-c series; FIG. S1 B presents in silico screen identified 23-compounds with potential binding properties to site 5 of HIF2a PAS-A. They are labelled as Compounds-i series; FIG. S1C presents 786-0 cells were treated with 10pM of each compound generated for the PAS-B pocket of HIF2a (c1-23) for 24hrs. Cell viability was assessed by MTT assay. DMSO treated (vehicle) used as a control. Error bars represent the standard deviation (S.D.) of three independent experiments; FIG. S1D presents 786-0 cells that were treated with lOpM of each compound generated for the PAS-A pocket of HIF2a (i 1 -23) for 24hrs. Cell viability was assessed by MTT assay. DMSO treated (vehicle) used as a control. Error bars represent the standard deviation (S.D.) of three independent experiments; FIG. S1 E presents HEK293, Caki-1 , and 786-0 cells that were treated with 10pM of compounds i15, i19, c2, and c15 for 24hrs. Cell viability was assessed by MTT assay. DMSO treated (vehicle) used as a control. Error bars represent the standard deviation (S.D.) of three independent experiments.

[0053] FIG. S2 presents the inhibitory effect of Compound-d 5 on ccRCC cell lines (Related to FIG. 2A-2H) VHL-null ccRCC cell lines (A498, 786-0), VHZ.-positive ccRCC cell line (Caki-1 ), and HEK293 cells were treated with the indicated amounts of Compound-ci 5 for 24hrs. The effect of Compound-d 5 on cell viability was assessed by MTT assay. Error bars represent the standard deviation (S.D.) of three independent experiments.

[0054] FIG. S3A-S3C present mechanism of Compound-c2 binding to HIF2a (as related to FIG. 3A-3E); FIG. S3A presents the synthesis of Compounds-v, x, w, y, z, and Biotin-Compound-c2 (10). Attachment of the alkyne to the quinazolinone benzamide was also explored and coupling of iodide (5) with Boc-propargyl amine was successful, providing alkyne (11 ), which could be deprotected with TFA to give the ammonium salt (12). Unfortunately, attempts to directly couple biotin to this ammonium salt was also hindered by the formation of complex mixtures, so the route utilizing theprotected biotin was utilized instead; FIG. S3B Wild-type (WT) HA-HIF2o and its mutants were transiently expressed in HEK293 cells and assessed by immunoblotting. GAPDH was used as a loading control; and FIG. S3C Lysates from (B) were challenged with 1 pM Biotin-Compound-c2 for 1 hr. HIF2a binding was assessed by immunoblotting.

[0055] FIG. S4 presents Compound-c2 does not target Hsp70 (as related to FIG. 5A-5H), wherein 786-0 cells were treated with 30pM Compound-c2 for the indicated times. Hsp70 protein levels were assessed by immunoblotting. GAPDH was used as a loading control.DETAILED DESCRIPTION OF THE INVENTION

[0056] The disclosed method and compositions may be understood more readily by reference the following detailed description of particular embodiments and the Example included therein and to the figures and their previous and following description.

[0057] Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those of ordinary skill in the art can easily carry out the present invention. However, the present invention may be implemented in several different forms and is not limited or restricted by the following examples.

[0058] In order to clearly explain the present invention, detailed descriptions of portions that are irrelevant to the description or related known technologies that may unnecessarily obscure the gist of the present invention have been omitted, and in the present specification, reference symbols are added to components in each drawing. In this case, the same or similar reference numerals are assigned to the same or similar elements throughout the specification.

[0059] Also, terms or words used in this specification and claims should not be restrictively interpreted as ordinary meanings or dictionary-based meanings, but rather should be interpreted consistently with the concepts disclosed in the present application, based on the principle that an inventor can act as his / her own lexicographer, defining terms so as to best describe and explain his or her invention.

[0060] Screening and identification of a novel HIF2a antagonist: The HIF2a-ARNT complex has a complicated architecture where ARNT spirals around the outside of each HIF subunit. This leads to a complex heterodimeric structure with five distinctsmall-molecule binding pockets (binding pockets 1-5) (FIG. 1A). These sites can be further subdivided into PAS domain encapsulated sites (labelled (1 , 2, 4, 5) in FIG. 1 A) as well as an interfacial cavity formed by the heterodimerizatrion of the two subunits (labeled 3 in FIG. 1A). HIF2a PAS-B domain (labelled (2) in FIG. 1A, FIG. 1B) has proven to be an attractive drug target. We therefore used this domain to conduct an in silico docking study using the previously solved X-ray crystal structure of the HIF2a- ARNT complex (PDB:4ZP4), (FIG. 1 A). We also performed docking at the HIF2a PAS- A domain (labelled (5) FIG. 1A, FIG. 1C). Since this domain has only 60% amino acid sequence identity with HIF2a PAS-B domain, it may provide selectivity towards structurally distinct inhibitors of HIF2a. A virtual screen with a library of drug-like compounds (-100,000 compounds, FIG. 1D) was performed using Autodock Vina employing MTiOpenScreen at sites 2 and 5 (FIG. 1A). This provided two sets of potential inhibitors, Compounds-c (from site 2, HIF2a PAS-B) and Compounds-i (from site 5, HIF2a PAS-A). From these, the top 23 compounds with the lowest predicted binding energies from each set were chosen for further evaluation (FIGs. S1A, S1B). We examined the effect of these 46-compounds at a single dose (10pM) for 24hrs on proliferation of ccRCC ( V7- / / _-null) 786-0 cells by the 3-(4, 5-dimethylthiazolyl-2)-2, 5- diphenyltetrazolium bromide (MTT) assay. These cells have elevated levels of HIF2a due to the lack of VHL gene. We set the inhibitory threshold to 60% viability compared to cells with treated with DMSO (vehicle control) and identified compounds c2 and c15 with the most effective impact on 786-0 cells (FIG. S1C). Since compounds i15 and i19 (targets of HIF2a PAS-A) showed modest inhibitory effect in 786-0 cells (FIG. S1 D), we decided to test them further alongside compounds c2 and c15 in control cell lines HEK293 and V / 7L-positive ccRCC cell line Caki-1 (FIG. S1E). Our data confirmed our earlier results that compounds-c2 and c15 have a -50% inhibitory effect on 786-0 cell proliferation (FIG. S1 E). Since Compounds-i15 and i19 did not reach this inhibitory threshold, we decided not to further examine them.

[0061] Compound-c2 mediated inhibition and proteasomal degradation of HIF2a: To examine the efficacy of our compounds in V77L-null ccRCC cells that are dependent on HIF2a, we treated a second V7-f / _-null ccRCC cell line, A498, with Compound-c2 and c15. Our data showed that Compound-c2 inhibited the growth of both ccRCC cell lines 786-0 and A498 (FIG. 2A). Conversely, this compound had no effect on normal HEK293 cells or ccRCC cells with intact VHL (Caki-1 ), suggesting its specificity for cells which are V / 7L-null and have high HIF2a levels (FIG. 2A). In contrast, Compound-c15 had growth inhibitory effect on our tested cell lines irrespective of presence or absence of the VHL gene, suggesting Compound-c15 may have off target effects in cells (FIG. S2). We therefore decided to further analyze only Compound-c2.

[0062] We next treated 786-0 cells with Compound-c2 and observed a marked increase in cleaved caspase-3 and cleaved caspase-7 (an indication of apoptosis) (FIG. 2B). To determine the effect of Compound-c2 on HIF2a function, we examined HIF2a activity by VEGF-luciferase reporter assay. We found that treatment of 786-0 cells with 30pM Compound-c2 led to significant downregulation of HIF2a activity (FIG. 20). We further showed that Compound-c2 also downregulates VEGF mRNA expression in a dose dependent manner (FIG. 2D). Interestingly, treatment of 786-0 cells with different amounts of Compound-c2 for 24hrs reduced levels of HIF2a (FIG. 2E). We further showed that diminished levels of HIF2a occurs with 4hrs treatment of 786-0 (FIG. 2F) and another VHL-null ccRCC cell line, OS-RC-2 (FIG. 2G). To determine if Compound-c2 caused HIF2a degradation via the proteosome, we cotreated 786-0 cells with Compound-c2 and the proteasome inhibitor Bortezomib and demonstrated stabilization of HIF2a protein level in these cells even in the presence of Compound-c2 (FIG. 2H). Taken together, our data demonstrates that Compound- c2 treatment inhibits HIF2a and leads to its proteasomal degradation.

[0063] Specificity and Selectivity of Compound-c2 for HIF2cc HIF exists in two main isoforms: HIF1a and HIF2a. In ccRCC, HIF1a has been shown to have tumor suppressor properties, while HIF2a acts to promote tumor growth. To determine whether Compound-c2 is specific for HIF2a, we first treated Caki-2 cells, which only express HIF1a, with Compound-c2. We found that Compound-c2 did not induce degradation of HIF1a (FIG. 3A).

[0064] To provide further support that Compound-c2 is binding HIF2a, a biotinylated version of Compound-c2 was developed (FIG. S3A). This involved the coupling of an alkyne tagged biotin to an iodinated version of Compound-c2 (FIG. S3A). Different amounts of Biotin-Compound-c2 (10) (FIG. S3A) were then used to challenge protein lysate from 786-0 cells. Our data showed HIF2a binding to 100nM Biotin-Compound-c2 (FIG. 3B). Additionally, we confirmed our observation by transiently expressing HA-HIF2o in HEK293 cells and demonstrated binding of HIF2o to 1 .OpM Biotin-Compound-c2 (FIG. 3C). We identified the amino acids that potentially contact Compound-c2 within the HIF2a PAS-B pocket and mutated each residue (F244A, S246A H248A, M252A, F254A, Y281A, Y307A, M309A, T321A, I337A,C339A and N341A) (FIG. 3D). We also mutated residues within the PAS-B pocket known to contribute to PT2399 binding (A277E, F280A) to verify that Compound-c2 to has a distinct binding mechanism to HIF2a. These mutants were transiently expressed in HEK293 cells and their lysate were challenged by Biotin-Compound-c2. Our data showed that all mutants listed above disrupted binding to Compound-c2, with an exception of HIF2a- F244A, A277E, and Y281A (FIGs. 3E, S3B, and S3C). Taken together, our data confirms the specificity of Compound-c2 for the PAS-B pocket of HIF2a.

[0065] Identification of the Compound-c2 pharmacophore: To identify the pharmacophore of Compound-c2, we tested some of the intermediate compounds that were prepared during the incorporation of biotin (FIG. S3A) for biological activity. Intermediates 1 (Compound-v), 3 (Compound-w), 2 (Compound-x), 4 (Compound-y), and 11 (Compound-z) (FIG. 4A, and FIG. S3A) were evaluated by treating 786-0 cells at 10pM for 24hrs (FIG. 4B). Cells were then assessed for induction of apoptosis by immunoblotting. Our data showed that only Compound-y and Compound-z caused marked induction of apoptosis, as well as a reduction of HIF2a levels (FIG. 4B). This indicates that both the quinazolinone and the benzamide groups are necessary for inducing apoptosis in 786-0 cells. We found a similar induction of the apoptotic marker cleaved-PARP in a second V77L-nul I ccRCC cell line, A498, treated with Compound-y and Compound-z, with a reduction in HIF2a levels similar to that caused by Compound-c2 treatment (FIG. 4C).

[0066] To examine the specificity of Compound-y and Compound-z for HIF2a, we tested the effect of these compounds for 24hrs on the proliferation of VHL-nu\\ ccRCC cells (786-0, A498) and V7- / L-positive ccRCC cells (Caki-1 ) by (MTT) assay with HK- 2 (normal renal proximal tubule cells), cells as a control. Our data showed that Compound-y had a growth inhibitory effect on our tested cell lines irrespective of presence or absence of the VHL gene, suggesting Compound-y may have off target effects in cells (FIG. 4D). In contrast, Compound-z inhibited the growth of both ccRCC cell lines 786-0 and A498, but had no effect in ccRCC cells with intact VHL (Caki-1 ) or normal HK-2 cells, suggesting its specificity for cells which are W- / / _-null and have high HIF2a levels (FIG. 4E). These findings suggest that the substitution pattern on the benzamide is critical to specific inhibition of HIF2o. Based on this information, we have modeled the predicted binding of Compound-z to the HIF2a PAS-B pocket (FIG. 4F). Collectively, our data demonstrated that the two intermediates of Biotin-Compound-c2 (Compounds-y and -z) have biological activity in V7- / L-null ccRCC cells; however, only Compound-z is specific toward HIF2a.

[0067] Compound-c2 disrupts HspZO chaperoning of HIF2a: Compound-c2 leads to proteasomal degradation of HIF2a, even in the absence of the VHL-E3 ubiquitin ligase. Therefore, this raised the possibility of a chaperone-dependent pathway for the elimination of HIF2a. The molecular chaperone Hsp70 is involved in the folding and stability of many proteins also known as clients. Here, we provide evidence that HIF2a also binds to Hsp70 by immunoprecipitating endogenous HIF2ct and co- immunoprecipitating Hsp70 from 786-0 cells (FIG. 5A). We next transiently coexpressed HA-HIF2a with different FLAG tagged domains of Hsp70, nucleotide binding domain (NTB) and substrate binding domain (SBD), in HEK293 cells (FIG. 5B). Immunoprecipitation of FLAG-Hsp70-SBD appears to preferentially co- immunoprecipitate HA-HIF2a, suggesting that HIF2a is a client of Hsp70 (FIG. 5B). To evaluate the effect of Hsp70 activity on HIF2a, we treated 786-0 cells with the Hsp70 inhibitor, JG98. Inhibition of Hsp70 led to the degradation of HIF2a (FIG. 5C), where co-treatment with Bortezomib stabilized HIF2a level (FIG. 5D), suggesting HIF2a relies on Hsp70 for its stability and activity. It is noteworthy that Compound-c2 does not lead to changes in Hsp70 protein levels (FIG. S4). Since both the disruption of the Hsp70:HIF2a complex by the Hsp70 inhibitor JG98 and treatment with Compound-c2 results in HIF2a degradation, we asked if Compound-c2 disrupts its interaction with Hsp70. Our data showed that immunoprecipitation of endogenous HIF2a followed by brief (2hrs) treatment of 780-0 cells with Compound-c2 caused dissociation of the HIF2a:Hsp70 complex (FIG. 5E). Additionally, we tested the effect of intermediate Compounds-y and -z to toward HIF2a and Hsp70 binding. Our results confirmed that treatment with both Compound-y and Compound-z disrupted the Hsp70:HIF2a complex (FIG. 5F).

[0068] Our earlier data (FIG. 3E) suggested that HIF2a-S246A, or H248A, or F280A mutants had reduced affinity towards Biotin-Compound-c2. As an alternative approach for targeting these mutants, we tested whether they are sensitive to Hsp70 inhibition. Our data indicates that treatment of HEK293 cells transiently expressing the above mutants with 10pM of JG98 led to degradation of HIF2a (FIG. 5G). Taken together, our data suggest that HIF2a is a client of the molecular chaperone Hsp70. Compound-c2 and Compound-z specifically binds to HIF2a and disrupts its interaction with Hsp70 to cause its proteasomal degradation (FIG. 5H).

[0069] Belzutifan is an FDA approved HIF2a inhibitor for patients with advanced renal cell carcinoma (RCC) following treatment with a programmed death receptor-1 (PD-1 ) or programmed death-ligand 1 (PD-L1 ) inhibitor and a vascular endothelial growth factor tyrosine kinase inhibitor (VEGF-TKI). This achievement was based on our deep understanding of structure and function of HIF2a, which led to the identification and further development of small molecule HIF2a inhibitors. Therefore, targeting HIF2a through pharmacologic inhibition offers a novel therapeutic strategy for HIF2a-driven cancers. Here, we utilized the crystal structure of HIF2ct and an in silica drug screening approach to identify new HIF2a inhibitors. A library of small druglike molecules were docked in both the PAS-A and PAS-B pockets. This approach led to the identification of Compound-c2 that specifically binds to the PAS-B pocket of HIF2a. Our data based on mutagenesis of HIF2a suggests that Compound-c2 makes contacts with 11 residues in the PAS-B pocket. Given the pro-survival role of HIF2a in t / HL-null ccRCC, Compound-c2 caused apoptosis only in ccRCC cell lines that lacked the VHL gene. Our data, based on binding and pharmacologic inhibition of Hsp70 in V7- / L-null ccRCC cells, suggested that indeed HIF2a is a client of Hsp70. Therefore, we have provided a model in which Compound-c2 binds to HIF2a PAS-B pocket, causing the dissociation of HIF2a from Hsp70 and its consequent ubiquitination and degradation in the proteasome. Further analysis of small molecules based on Compound-c2 suggested that the quinazolinone and the benzamide groups provided the pro-apoptotic activity of these molecules, with the substitution on the benzamide being critical to selective HIF2a inhibition. These components can potentially serve as attractive scaffolds for the design and synthesis of compounds similar to Compound-c2 with improved binding affinity.

[0070] Targeted therapy by any small molecules in cancer eventually leads to acquired drug resistance. One mechanism of acquired resistance is mutation of the drug target. Previous studies in Patient-Derived Xenograft (PDX) models have demonstrated that prolonged exposure of ccRCC tumors to PT2385 (precursor to Belzutifan) acquired resistance as the result of a mutation (G323E) in HIF2a. Although G323 is in the HIF2a PAS-B domain, Compound-c2 does not make contact with the G323 residue. Therefore, it can be potentially used to combat tumor resistance to Belzutifan. Furthermore, the Compound-c2 mode of action that results in inhibition of HIF2a appears to be different from Belzutifan and its analogs. Compound-c2 binding to HIF2a PAS-B caused its dissociation from the molecular chaperone Hsp70. Thisled to potential ubiquitination of HIF2a, which is independent of the VHL ubiquitination machinery and ultimate degradation in the proteasome (FIG. 5H). Our findings also showed that HIF2a is indeed a client of Hsp70 and it preferentially binds to the substrate binding domain of Hsp70. This suggests that perhaps in the presence of oxygen, HIF2a must initially dissociate from the molecular chaperone Hsp70 and this allows for prolyl-hydroxylation and VHL mediated ubiquitination and degradation of HIF2a. This also raises the possibility of the involvement of other molecular chaperones such as Hsp90 towards HIF2ct stability. Previous work has shown the involvement of Hsp90 in the chaperoning of HIF1a. Therefore, targeting the molecular chaperones, and consequently activating the VHL-independent HIF2a -degradative pathway, may be an attractive therapeutic approach for the treatment of ccRCC.

[0071] The following are provided for exemplification purposes only and are not intended to limit the scope of the embodiments described in broad terms above.

[0072] Cell lines: Cultured human embryonic kidney (HEK293), (Human, unknown) and HK-2 (Human, Male) cells were grown in Dulbecco’s Modified Eagle Medium (DMEM, Sigma-Aldrich), 786-0 cells (Human, Male) OS-RC-2 cells (Human, Male) in Roswell Park Memorial Institute (RPMI)1640 Medium (Sigma-Aldrich), A498 cells (Human, Female) in Minimum Essential Medium (MEM, Sigma-Aldrich), Caki-1 (Human, Male) and Caki-2 cells (Human, Male) in McCoy’s 5A Medium (Sigma- Aldrich) all supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich). Cells were maintained in a CellQ incubator (Panasonic Healthcare) at 37°C in an atmosphere containing 5% CO2.

[0073] Plasmids: For mammalian expression HA-HIF2alpha-pcDNA3 was a gift from William Kaelin (Addgene plasmid # 18950; / / n2t.net / addgene: 18950; RRID:Addgene_18950) and point mutations F244A, H248A, M252A, F254A, Y281A, Y307A, M309A, T321A, and N341A, S246A, A277E, F280A, I337A, and C339A were created by site-directed mutagenesis (see Primer Table FIG. 6) and confirmed by DNA sequencing. The pcDNA3-FLAG-Hsp70 and sub-domains (NTB and SBD) were cloned using primers in FIG. 6 Primer Table.

[0074] Cell Transfection and Treatment: Cultured cells were split and then transfected the following day when about 40% confluent with each construct using Mirus TranslT-2020 (MirusBio) according to manufacturer’s protocol. Cells were incubated at 37°C and then extracted or collected for analysis after 24hrs (HEK293). Short interfering RNA (siRNA) scramble control and EPAS1 (HIF2a) targetingduplexes were purchased from OriGene (SKU: SR320042A, SR320042B, and SR320042C). Indicated cells were transiently transfected with the siRNA using Mirus TranslT-2020. For HI F2a knock-down, either 30nM of control siRNA or 10nM of each HIF2a siRNA duplex (A, B and C) were mixed prior to transfection. Cells were incubated at 37°C for 72hrs, then harvested for protein extraction. Cells were then harvested for protein extraction.

[0075] Protein Extraction, Immunoprecipitation, and Immunoblotting: Protein extraction from mammalian cells was carried out using methods previously described. Cell lysates were quantified using 1X Bradford reagent (BioRad). For immunoprecipitation, cell lysates were incubated with anti-FLAG antibody conjugated beads (Sigma) at 4°C for 2hrs. Endogenous IPs were achieved by incubating lysate with anti-HIF2a antibody (Cell Signaling), 2hrs followed by protein G agarose (Invitrogen) at 4°C for 2hrs. Immunopellets were washed 4 times with fresh lysis buffer (20mM Tris-HCI (pH 7.4), 100 mM NaCI, 1 mM MgCI2, 0.1 % NP40, protease inhibitor cocktail (Roche), and PhosSTOP (Roche)) and eluted in 5x Laemmli buffer. Precipitated proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Co-immunoprecipitated proteins or proteins from cell lysate were detected with antibodies stated in the Key Resources Table.

[0076] Cell Viability Assay: Renal cancer cell lines 786-0, Caki-1 , A498 cells as well as HEK293 and HK-2 cells were plated at 10,000 cells per well in 96-well plates. Cells were treated with different amounts of inhibitors and DMSO was used as control. After 24hrs, cell viability assay was performed using the Quick Cell Proliferation Kit Plus (BioVision) according to the manufacturer’s protocol. The absorbance at 450nm was measured on a Tecan Infinite M200 Pro and proliferation rate was calculated.

[0077] In silico Docking: Virtual screening was conducted with a library of drug like compounds (diverse-lib, -100,000 compounds, FIG. 1 D). This was performed using Autodock Vina on the MTiOpenScreen server.

[0078] qPCR: RNA was extracted using RNA extraction mini-kit and quantified using NanoQuant (TECAN). RNA (1mg) from each sample was reverse transcribed into cDNA using iScript cDNA synthesis kit (BioRad) as previously described29. RT- qPCR analysis was performed using nucleic acid stain SYBR-GREEN (BioRad) and specific qPCR oligonucleotide primers: VEGF1 and cyclophilin control. VEGF mRNA levels were normalized to cyclophilin control.

[0079] VEGF-luciferase reporter: 786-0 cells were transiently transfected with VEGF-luciferase reporter for 24hrs as previously described. Cells were plated in a 96- well plate for 24hrs followed by treatment with 30pM of Compound-c2 for an additional 8hrs. siRNA targeting HIF2a was used as a control. One-Gio Luciferase reagent was added for 30 minutes and luminescence was read using Tecan SPARK. Data was normalized to DMSO control.

[0080] Synthesis of Biotinylated-Compound-c2, (2-(3-Ethylureido)benzonitrile (2)): A mixture of 2-aminobenzonitrile 1 (5.0 g, 42.3 mmol) and ethyl isocyanate (3.35 mL, 42.3 mmol) were slightly heated to dissolve. The reaction mixture was stirred for 26 h under argon. The precipitate was recrystallized from MeOH to afford the urea product 2 (3.9 g, 48%) as a white fluffy solid. This compound has been previously reported.51TLC Rf = 0.38 (30% EA / hexanes);1H NMR (400 MHz, DMSO-ds) 3 8.43 (s, 1 H), 8.01 (d, J = 8.4 Hz, 1 H), 7.67 (d, J = 7.6 Hz, 1 H), 7.57 (t, J = 8.0 Hz, 1 H), 7.08 (t, J = 7.6 Hz, 1 H), 6.95 (t, J = 4.7 Hz, 1 H), 3.16-3.09 (m, 2H), 1.07 (t, J = 7.2 Hz, 3H); );13C NMR (100 MHz, DMSO-ds) 0 154.3, 142.8, 133.8, 132.9, 122.1 , 120.6, 117.0, 100.9, 34.0, 15.1.

[0081] 3,4-Dihydro-4-imino-3-ethylquinazolin-2(1 H)-one (3): The benzonitrile 2 (1.68 g, 8.87 mmol) was dissolved in EtOH (15 mL) with heating. Ammonium hydroxide (8.9 mL) was added and the reaction was refluxed for 1 h. The reaction mixture was cooled to rt and DI water (30 mL) was added. The precipitate was vacuum filtered, washed with DI water, and dried using an azeotrope of EtOH to yield the product 3 (1.48 g, 88%) as a white fluffy solid. This compound has been previously reported.51TLC Rf= 0.31 (100% EA);1H NMR (400 MHz, DMSO-ds) 6 10.68 (s, 1 H), 8.85 (s, 1 H), 8.05 (d, J = 8.0 Hz, 1 H), 7.46 (t, J = 8.4 Hz, 1 H), 7.09 (t, J = 7.6 Hz, 1 H), 7.03 (d, J = 8.4 Hz, 1 H), 4.06 (q, J = 6.18 Hz, 2H), 1 .14 (t, J = 6.18 Hz, 3H);13C NMR (100 MHz, DMSO-ds) 6 155.2, 149.8, 136.9, 132.7, 125.9, 122.0, 115.1 , 113.0, 35.7, 12.3.

[0082] 4-Bromo-N-[3-ethyl-2-oxo-2,3-dihydro-4(1H)quinazolinyliden]benzenecarboxamide (y): NaH (2 equiv.) was suspended in dry DMA and stirred for 15 min. A solution of quinazoline 3 (1 equiv.) dissolved in dry DMA (0.1 M) was added dropwise to the reaction, followed by addition of the corresponding benzoyl chloride (1.5 equiv). The reaction was stirred for 24 h. EA and DI water were added and the layers were separated. The aqueous layer was extracted with EA (2x). The combined organic layers were washed with DI water (2x) and 2% LiCI, dried overNazSCU, filtered, and concentrated under vacuum. The residue was purified via silica gel chromatography (50% EA / hexanes) to yield the bromide 4 (393 mg, 21%) as a white solid; mp = 142-145 ”C; TLC Rf= 0.54 (50% EA / hexanes); IR (ATR) 3304, 3284, 2978, 1713, 1681 , 1163, 673 cm-1;1H NMR (400 MHz, CDCh) 6 7.93 (d, J = 7.5 Hz, 1 H), 7.80 (d, J = 9.1 Hz, 2H), 7.64 (d, J = 8.6, 2H), 7.45 (t, J = 8.3 Hz, 1 H), 7.26 (t, J = 8.0 Hz, 1 H), 6.97 (d, J = 8.1 Hz, 1 H), 4.19 (q, J = 14.0, 6.9 Hz, 2H), 1.30 (t, J = 7.0 Hz, 3H);13C NMR (100 MHz, CDCh) 3 170.2, 155.2, 149.0, 135.8, 133.5, 132.8, 131 .6, 130.6, 128.2, 126.4, 124.4, 115.2, 114.3, 37.7, 12.3; Anal. Calcd for CvHuBrNsCh: C, 54.86; H, 3.79; N, 11.29. Found: C, 54.56; H, 3.71 ; N, 11.20.

[0083] 4-lodo-A / -[3-ethyl-2-oxo-2,3-dihydro-4(1 H)- quinazolinyliden]benzenecarboxamide (5): NaH (2 equiv.) was suspended in dry DMA and stirred for 15 min. A solution of quinazoline 3 (1 equiv.) dissolved in dry DMA (0.1 M) was added dropwise to the reaction, followed by addition of the corresponding benzoyl chloride (1 .5 equiv.). The reaction was stirred for 24 h. Ethyl acetate (EA) and water were added and the layers were separated. The aqueous layer was extracted with EA (2x). The combined organic layers were washed with DI water (2x) and 2% LiCI, dried over Na2SC>4, filtered, and concentrated under vacuum. The residue was purified via silica gel chromatography (3% MeOH / DCM) to yield the iodide 5 (161 mg, 36%) as a white solid; mp = 160-163 °C; TLC Rf= 0.40 (1 % MeOH / DCM); IR (ATR) 3304, 3285, 2978, 1712, 1670, 1471 , 598 cm’1;1H NMR (400 MHz, CD3CN) 0 8.88 (s, 1 H), 7.73 (d, J = 8.4 Hz, 1 H), 7.42 (d, J = 8.8 Hz, 2H), 7.18 (d, J = 8.0 Hz, 2H), 6.92 (t, J = 7.6 Hz, 1 H), 6.73 (t, J = 6.8 Hz, 1 H), 6.32 (d, J = 7.6 Hz, 1 H), 3.51-3.50 (m, 2H), 0.60 (t, J = 7.2 Hz, 3H);13C NMR (100 MHz, DMSO-ds) 5 170.8, 153.6, 138.5, 137.1 , 134.9, 133.1 , 131.9, 131.7, 129.0, 126.9, 123.8, 114.5, 104.7, 36.5, 12.1 ; Anal. Gated for Ci7Hi4lN3O2: C, 48.71 ; H, 3.37; N, 10.02. Found: C, 49.06; H, 3.39; N, 9.81 ; HRMS (ESI) m / z calcd for C17H15IN3O2 [M + H]+420.0203, found: 420.0201.

[0084] N-(Prop-2-ynyl)-d-biotinamide (7): Propargylamine (0.34 mL, 5.32 mmol) and N-methylmorpholine (0.59 mL, 5.32 mmol) were dissolved in dry DMF (15 mL) at 0 °C and stirred for 30 min under argon. EDC (826 mg, 5.32 mmol) and d-biotin 5 (1 .0 g, 4.09 mmol) were added and the reaction was stirred for 2 h and then warmed to rt and stirred for 24 h. The DMF was distilled off and the residue was purified via silica gel chromatography (15% MeOH / CHCh) to afford the product 7 (694 mg, 60%) as a white solid, mp = 162-165 °C; TLC Rf= 0.27 (10% MeOH / CHCh); IR (ATR) 3422, 3270, 2929, 2860, 1693, 1637, 1538, 1464 cm'1;1H NMR (400 MHz, DMSO-d6) 68.21(t, J = 5.2 Hz, 1 H), 6.41 (s, 1H), 6.35 (s, 1H), 4.32-4.28 (m, 1 H), 4.14-4.11 (m, 1H), 3.83 (dd, J = 5.4, 2.4 Hz, 2H), 3.12-3.07 (m, 2H), 2.82 (dd, J = 12.8, 4.9 Hz, 1 H), 2.57 (d, J = 12.0 Hz, 1H), 2.08 (t, J = 7.0 Hz, 2H), 1.65-1.24 (m, 6H);13C NMR (100 MHz, DMSO-de) 6 171.8, 162.7, 81.3, 72.8, 61.0, 59.2, 55.4, 39.8, 34.8, 28.1 , 28.0, 27.7, 25.1 ; Anal. Calcd for C13H19N3O2S: C, 55.49; H, 6.81 ; N, 14.93. Found: C, 55.62; H, 6.87; N, 14.68.

[0085] 1 ’N-(4,4’-Dimethoxytrityl)-11 ’N-(prop-2-ynyl)-d-biotinamide (8): Biotin amide7 (519 mg, 1.84 mmol) was dissolved in dry pyridine (10 ml_). DMTrCI (750 mg, 2.21 mmol) and DMAP (23 mg, 0.184 mmol) were added and the reaction was stirred under argon at 40 °C for 24 h. Methanol was added (5 mL) and stirred for 5 min under argon. The reaction mixture was concentrated under vacuum and the residue was dissolved in DCM (40 mL) and washed with NaHCOs (50 mL). The aqueous layer was extracted with DCM (3 x 20 mL). The organic layers were collected, washed with brine (40 mL), dried over NazSCU, and concentrated under vacuum. The crude residue was purified via silica gel chromatography (1% MeOH / CHCh to 4% MeOH / CHCh) to afford the product 8 (730 mg, 68%) as a white foam, mp = 87-90 °C; TLC Rf = 0.27 (4% MeOH / CHCh); IR (ATR) 3420, 3271 , 3066, 2929, 2860, 1692, 1647, 1507, 1249 cnr1;1H NMR (400 MHz, CD3OD) 5 7.88 (s, 1 H), 7.29-7.22 (m, 5H), 7.17-7.11 (m, 4H), 6.82 (d, J = 8.8 Hz, 4H), 4.54-4.50 (m, 1 H), 4.37-4.34 (m, 1 H), 3.92 (d, J = 2.5 Hz, 2H), 3.76 (s, 6H), 3.16-3.12 (m, 1 H), 2.56 (t, J = 2.4 Hz, 1 H), 2.31 (d, J = 13.6 Hz, 1H), 2.24 (dd, J = 12.8, 5.5 Hz, 1 H), 2.17 (t, J = 7.2 Hz, 2H), 1.77-1.53 (m, 4H), 1.45-1.37 (m, 2H);13C NMR (100 MHz, DMSO-de) 6 171 .8, 160.8, 157.8, 141.1 , 135.9, 131.0, 129.4, 127.3, 112.6, 81 .4, 79.2, 72.8, 71 .8, 64.5, 59.3, 55.0, 54.3, 38.6, 34.9, 28.4, 28.1 , 27.7, 25.1 ; Anal. Calcd for C34H37N3O4S: C, 69.96; H, 6.39; N, 7.20. Found: C, 69.58; H, 6.39; N, 6.83.

[0086] TN-(4,4’-Dimethoxytrityl)-N-(prop-1-ynyl)-d-biotinamide-N-(3-ethyl-2-oxo- 1,2,3,4-tetrahydroquinazolin-4-ylidene)benzamide (9): Pd(PPh3)2Cl2 (12 mg, 0.0171 mmol), Cui (7 mg, 0.0343 mmol), biotin amide 8 (100 mg, 0.171 mmol) and iodide 5 (86 mg, 0.206 mmol) were added to a round bottom flask and purged with argon. Dry DMA (1 .7 mL) and dry TEA (3.4 mL) were added and the reaction mixture was stirred for 24 h under argon. DI water (10 mL) and EA (10 mL) were added and the layers were separated. The organic layer was washed with DI water (2 x 10 mL), dried over Na2SO4, and concentrated under vacuum. The crude residue was purified via silica gel chromatography (5% MeOH / DCM) to give the coupled product 9 (83mg, 55%) asa pale yellow solid, mp = 148-150 °C; TLC Rf= 0.49 (10% MeOH / CHCb); IR (ATR) 2980, 2359, 1676, 1603, 1507, 1245 erm1;1H NMR (400 MHz, Acetone-d6) 0 8.21 (d, J = 7.3 Hz, 1 H), 8.01 (d, J = 8.4 Hz, 2H), 7.57 (d, J = 8.4 Hz, 2H), 7.52-7.44 (m, 2H), 7.35-7.23 (m, 6H), 7.16 (t, J = 9.2 Hz, 4H), 6.96 (d, J = 8.4 Hz, 1 H), 6.84 (d, J = 8.0 Hz, 4H), 6.03 (bs, 1 H), 4.48-4.38 (m, 2H), 4.22 (d, J = 4.2 Hz, 2H), 4.15 (q, J = 6.5 Hz, 2H), 3.78 (s, 6H), 3.20-3.16 (m, 1 H), 2.81 (bs, 1H), 2.36 (d, J = 11 .1 Hz, 1 H), 2.28 (dd, J = 12.7, 5.3 Hz, 1H), 2.17-2.13 (m, 2H), 1.78-1.36 (m, 6H), 1.22 (t, J = 6.7 Hz, 3H);13C NMR (100 MHz, DMSO-de) 6 172.7, 169.9, 161.8, 158.4, 155.1 , 148.8, 143.8,135.8, 135.7, 133.5, 132.5, 131.3, 130.1 , 129.7, 127.6, 127.0, 126.3, 124.2, 115.0,112.8, 90.6, 81.9, 72.8, 65.4, 59.7, 55.2, 54.4, 39.3, 37.6, 35.2, 31.6, 29.7, 28.2, 27.8, 25.0, 22.7, 14.1 , 12.1 ; HRMS (ESI) m / z calcd for CsiHsoNeOeSNa [M +Na]+897.3405, found: 897.3406.

[0087] Biotin-Compound-c2 (10); Protected biotin conjugate 9 (15 mg, 0.131 mmol) was dissolved in 3% TFA in DCM (1 ml_). The reaction mixture was stirred for 2 h. MeOH (3 mL) was added and the mixture was concentrated under vacuum. The crude residue was purified via silica gel chromatography (5% MeOH / DCM to 4% MeOH; 95% DCM: 1 % TEA) to yield the Biotin-Compound-c2 conjugate 10 (46 mg, 61 %) as a pale yellow solid, mp = 159-162 °C; TLC Rf= 0.63 (20% MeOH / CHCb); IR (ATR) 2926, 2019, 1701 , 1602, 1232 cm’1;1H NMR (400 MHz, DMSO-de) 6 9.45 (bs, 1 H), 8.37 (t, J = 5.3 Hz, 1 H), 8.29 (d, J = 7.9 Hz, 1 H), 7.98 (d, J = 8.2 Hz, 2H), 7.58 (d, J = 8.2 Hz, 2H), 7.50 (t, J = 7.9 Hz, 1 H), 7.29 (t, J = 7.9 Hz, 1 H), 6.87 (d, J = 8.2 Hz, 1 H), 6.41 (s, 1 H), 6.34 (s, 1 H), 4.29-4.26 (m, 1 H), 4.15 (d, J = 5.2 Hz, 2H), 4.12-4.05 (m, 3H), 3.10- 3.05 (m, 1H), 2.79 (dd, J = 13.1 , 5.4 Hz, 1 H), 2.55 (d, J = 12.4 Hz, 1 H), 2.12 (t, J = 7.7 Hz, 2H), 1.65-1.29 (m, 6H), 1.16 (t, J = 6.2 Hz, 3H);13C NMR (100 MHz, DMSO-de) 6 172.4, 172.3, 170.4, 163.0, 148.7, 135.2, 133.6, 132.5, 131.9, 130.8, 129.3, 127.2, 124.2, 114.6, 114.1 , 92.5, 80.7, 61.3, 59.4, 55.6, 36.8, 35.1 , 28.8, 28.7, 28.3, 28.2, 25.3, 12.2; HRMS (ESI) m / z calcd for C31H32N6O4S [M + H]+573.2279, found: 573.2272.

[0088] tert-Butyl N-[3-(4-{[(4E)-3-ethyl-2-oxo-1 ,2,3,4-tetrahydroquinazolin-4- ylidene] carbamoyl} phenyl)prop-2-yn-1-yl]carbamate (11 ): Boc protected propargyl amine (15 mg, 0.099 mmol), iodide (5) (50 mg, 0.119 mmol), Pd(PPh3)2Cl2 (7 mg, 0.010 mmol), and Cui (4 mg, 0.019 mmol) were purged with argon. Dry THF (0.9 mL) and dry TEA (2 mL) were added and the reaction was stirred for 24 h under argon. DI water (5 mL) and EA (3 mL) were then added. The aqueous layer wasextracted with EA (2 x 3 ml_). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, and concentrated under vacuum. The crude residue was purified via silica gel chromatography (40% EA / hexanes) to afford the alkyne 11 (35 mg, 81 %) as a yellow solid, mp = 135-137 °C; TLC Rf = 0.11 (40% EA / hexanes); IR (ATR, cm’1) 2980, 2932, 2168, 1672, 1162;1H NMR (400 MHz, acetone-ds) 6 8.94 (bs, 1 H), 8.21 (d, J = 8.4 Hz, 1 H), 8.02 (d, J = 7.8 Hz, 2H), 7.58 (d, J = 7.8 Hz, 2H), 7.51 (t, J = 7.8 Hz, 1 H), 7.29 (t, J = 7.8 Hz, 1 H), 6.97 (d, J = 8.4 Hz, 1 H), 6.48 (bs, 1 H), 4.19-4.14 (m, 4H), 1.43 (s, 9H), 1.23 (t, J = 6.8 Hz, 3H);13C NMR (100 MHz, acetone-de) 6 171.3, 156.3, 154.9, 149.9, 136.5, 133.8, 133.5, 133.0, 131.2, 130.4, 127.4, 124.6, 115.6, 115.5, 92.6, 81.7, 79.4, 37.5, 31.3, 28.6, 12.5; HRMS (ESI) m / z calcd for C25H26N4O4Na [M + Na]+469.1846, found: 469.1846.

[0089] 3-(4-{[(4E)-3-ethyl-2-oxo-1 ,2,3,4-tetrahydroquinazolin-4- ylidene]carbomoyl) phenyl)prop-2-yn-1 -ammonium trifluoroacetate (12):The Boc protected amine 11 (43 mg, 0.0963 mmol) was dissolved in TEA (1 mL) and stirred for 2 h. The reaction mixture was then concentrated under vacuum. The residue was dissolved in MeOH (3 x 10 mL) and then concentrated, followed by DCM (3 x 10 mL) to yield the TEA salt 12 (44 mg, 100%) as a light brown solid, mp = 98-101 °C; TLC Rr = 0.38 (10% MeOH / DCM); IR (ATR) 2996, 1973, 1669, 1601 , 1129 cm'1;1H NMR (400 MHz, CD3OD) 6 8.43 (d, J = 8.4 Hz, 1 H), 8.13 (d, J = 8.42 Hz, 2H), 7.86 (t, J = 8.0 Hz, 1 H), 7.68 (d, J = 8.4 Hz, 2H), 7.56 (t, J = 8.0 Hz, 1 H), 7.19 (d, J = 8.4 Hz, 1 H), 4.25 (q, J = 7.0 Hz, 2H), 4.10 (s, 2H), 1.38 (t, J = 7.3 Hz, 3H);13C NMR (100 MHz, CD3OD) 6 169.0, 160.3, 147.5, 139.4, 139.0, 133.8, 132.8, 132.2, 130.4, 127.7, 126.6, 116.7, 110.4, 86.7, 86.3, 41.3, 30.7, 11.5; HRMS (ESI) m / z calcd for C2OHI9N402+[M]+347.1503, found: 347.1501.

[0090] Biotin-Compound-c2 pulldown: HEK293 cells were transiently transfected with HA-HIF2a or active site point mutants and protein lysate extracted. Lysate was incubated with indicated amounts of Biotin-Compound-c2 (10) at 4°C for 1 hr then added to streptavidin-conjugated agarose and incubated at 4°C for 1 hr. Following three washes with fresh extraction buffer, bound proteins were eluted in 5x Laemmli buffer. Samples were run by SDS PAGE, transferred to nitrocellulose membrane, and analyzed by Western blot.

[0091] Quantification and Statistical Analysis: Presented data are representative of three biological replicates, unless otherwise specified. All statistical analysis were performed using GraphPad Prism version 9.5.0 for Windows (GraphPad Software).Statistical significance was ascertained between individual samples using a parametric unpaired f-test or Tukey's multiple comparisons test where indicated. Significance is denoted by asterisks in each figure: *P < 0.05; **P < 0.01 ; ***P < 0.001 ; ****p < 0.0001. Error bars represent the standard deviation for three independent experiments, unless otherwise indicated.

[0092] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein can be different from the actual publication dates, which can require independent confirmation.

[0093] All patents, patent applications, and other scientific or technical writings referred to anywhere herein are incorporated by reference herein in their entirety. The embodiments illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that are specifically or not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising," "consisting essentially of," and “consisting of” can be replaced with either of the other two terms, while retaining their ordinary meanings. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it Is recognized that various modifications are possible within the scope of the claims. Thus, it should be understood that although the present methods and compositions have been specifically disclosed by embodiments and optional features, modifications and variations of the concepts herein disclosed can be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of the compositions and methods as defined by the description and the appended claims.

[0094] Any single term, single element, single phrase, group of terms, group of phrases, or group of elements described herein can each be specifically excluded from the claims.

[0095] Whenever a range is given in the specification, for exampie, a temperature range, a time range, a composition, or concentration range, all intermediate ranges and subranges, as well as ail individual values included in the ranges given are intended to be included in the disclosure. It will be understood that any subranges or individual values in a range or subrange that are included in the description herein can be excluded from the aspects herein. It will be understood that any elements or steps that are included in the description herein can be excluded from the claimed compositions or methods

[0096] In addition, where features or aspects of the compositions and methods are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the compositions and methods are also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.REFERENCES

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Claims

What We Claim Is:1 . A method of inhibiting Hypoxia-Inducible Factor 2 alpha (HIF2a) in a patient in need thereof, said method comprising administering a therapeutically effective amount of at least one Hypoxia-Inducible Factor 2 alpha (HIF2a) antagonist compound having the structural formula I:wherein X is a halogen, an alkyl, a vinyl, an alkynyl or an aryl; Y is a halogen, an alkyl, a vinyl, an alkynyl or an aryl; R1is an alkyl or an aryl; and R2is an alkyl or an aryl, or a pharmaceutically acceptable salt thereof.

2. The method of inhibiting HIF2a of claim 1 , wherein the HIF2a antagonist compound is least one selected from the group consisting of:or a pharmaceutically acceptable salt thereof.

3. The method of inhibiting HIF2a of claim 1, wherein the HIF2a antagonist compound is at least one selected from the group consisting of: 3-ethyl-4-(p- fluorobenzoylimino)-3,4-dihydro-2(1 / 7)-quinazolinone, 4-bromo-N-[3-ethyl-2-oxo-2,3 dihydro4(1 H)quinazolinyliden]benzenecarboxamide), and tert-butyl N-[3-(4-{[(4E)-3- ethyl-2-oxo-1 ,2,3,4-tetrahydroquinazolin-4-ylidene] carbamoyl} phenyl)prop-2-yn-1- yl]carbamate), or a pharmaceutically acceptable salt thereof.

4. The method of inhibiting HIF2a of claim 1 , wherein the patient is further treated with one more additional therapies.

5. The method of inhibiting HIF2a of claim 4, wherein the one or more therapies comprise chemotherapy, cancer immunotherapy, surgical removal of all or part of the cancerous tissue, or radiation therapy.

6. A method of inhibiting HIF2a in a patient in need thereof, said method comprising administering a therapeutically effective amount of at least one HIF2a antagonist compound selected from the group consisting of:or a pharmaceutically acceptable salt thereof.

7. The method of inhibiting HIF2a of claim 6, wherein the HIF2a antagonist compound is at least one selected from the group consisting of: 2-aminobenzonitrile, 3,4-dihydro-4-imino-3-ethylquinazolin-2(1 H)-one, and 2-(3-ethylureido)benzonitrile, or a pharmaceutically acceptable salt thereof.

8. The method of inhibiting HIF2a of claim 6, wherein the patient is further treated with one more additional therapies.

9. The method of inhibiting HIF2a of claim 8, wherein the one or more therapies comprise chemotherapy, cancer immunotherapy, surgical removal of all or part of the cancerous tissue, or radiation therapy.

10. A Hypoxia-Inducible Factor 2 alpha (HIF2o) antagonist compound comprising the structural formula I:wherein X is a halogen, an alkyl, a vinyl, an alkynyl or an aryl; Y is a halogen, an alkyl, a vinyl, an alkynyl or an aryl; R1is an alkyl or an aryl; and R2is an alkyl or an aryl, or a pharmaceutically acceptable salt thereof.

11. The HIF2a antagonist compound of claim 10, wherein the compound at least one selected from the group consisting of: 3-ethyl-4-(p-fluorobenzoylimino)-3,4- dihydro-2(1 H)-quinazolinone, 4-bromo-N-[3-ethyl-2-oxo-2,3 dihydro4(1 H)quinazolinyliden]benzenecarboxamide), and tert-butyl N-[3-(4-{[(4E)-3- ethyl-2-oxo-1 ,2,3,4-tetrahydroquinazolin-4-ylidene] carbamoyl} phenyl)prop-2-yn-1- yl]carbamate), or a pharmaceutically acceptable salt thereof12. A Hypoxia-Inducible Factor 2 alpha (HIF2a) antagonist compound selected from the group consisting of:or a pharmaceutically acceptable salt thereof.

13. The Hypoxia-Inducible Factor 2 alpha (HIF2a) antagonist compound of claim 12, wherein the HIF2a antagonist compound is at least one selected from the group consisting of: 2-aminobenzonitrile, 3,4-dihydro-4-imino-3-ethylquinazolin-2(1 H)-one, and 2-(3-ethylureido)benzonitrile, or a pharmaceutically acceptable salt thereof.

14. The method of claim 5 or 9, wherein the therapy is treatment of clear cell renal cell carcinoma (ccRCC).

15. A method of proteasomal degradation of HIF2a in a patient in need thereof, comprising administering a therapeutically effective amount of at least one of the HIF2a antagonist compounds of claims 10 or 12, or a pharmaceutically acceptable salt thereof.

16. The method of claim 15, wherein the patient is further treated with one more additional therapies.

17. The method of claim 16, wherein the one or more therapies comprise chemotherapy, cancer immunotherapy, surgical removal of all or part of the cancerous tissue, or radiation therapy.

18. A method of reducing the interaction of HIF2a and a chaperone Heat shock protein-70 (Hsp70) in a patient in need thereof, comprising administering a therapeutically effective amount of at least one of the HIF2a antagonist compounds of claims 10 or 12, or a pharmaceutically acceptable salt thereof.

19. The method of claim 18, wherein the patient is further treated with one more additional therapies.

20. The method of claim 19, wherein the one or more therapies comprise chemotherapy, cancer immunotherapy, surgical removal of all or part of the cancerous tissue, or radiation therapy.

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