Direct-binding dual inhibitors of hypoxia-inducible factor 1 (HIF-1) and HIF-2

Abstract

Disclosed are direct-binding dual hypoxia-inducible factor (HIF) inhibitors and their use for treating cancer, including, but not limited to, breast, colorectal, lung, melanoma, pancreatic, and prostate cancer as a monotherapy and in combination with anti-CTLA-4 or anti-PD-1 immunotherapies. The dual HIF inhibitors also can be used for treating diseases, disorders, or conditions associated with ocular neovascularization, including, but not limited to, diabetic macular edema, diabetic retinopathy, and other ischemic retinopathies (including, but not limited to retinal vein occlusion, sickle cell retinopathy, retinopathy of prematurity, Norrie's disease, and Coat's disease), corneal neovascularization, and the treatment or prevention of neurovascular (wet-type) age-related macular degeneration.

Description

DIRECT-BINDING DUAL INHIBITORS OF HYPOXIA-INDUCIBLE FACTOR 1 (HIF-1) AND HIF-2 CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No.63 / 669,986 filed July 11, 2024, and U.S. Provisional Application No.63 / 663,363 filed June 24, 2024, each of which are incorporated herein by reference in its entirety. FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under grant GM131710 awarded by the National Institutes of Health. The government has certain rights in the invention. SEQUENCE LISTING

[0003] The text of the computer readable sequence listing filed herewith, titled “JHU_43011_601_SequenceListing.xml”, created June 24, 2025, having a file size of 46,819 bytes, is hereby incorporated by reference in its entirety. BACKGROUND

[0004] Hypoxia is defined as decreased O2 availability and is a common feature of many pathological conditions, including cancer, ischemic cardiovascular disease, and inflammatory disorders. Hypoxia-inducible factors (HIFs), including HIF-1α and HIF-2α, are transcriptional activators that mediate adaptation to decreased O2 availability. In the presence of normal O2 levels, HIF-1α and HIF-2α are subject to O2-dependent hydroxylation on two prolyl residues (Pro-402 and Pro-564 in human HIF-1α). The von Hippel-Lindau (VHL) tumor suppressor protein binds to the hydroxylated form of HIF-1α and HIF-2α, targeting them for ubiquitination and proteasomal degradation. Under hypoxic conditions, the hydroxylation reaction is inhibited and the non- hydroxylated forms of HIF-1α and HIF-2α, which are resistant to degradation, rapidly accumulate, dimerize with HIF-1β and bind to target genes to activate their transcription.

[0005] Pathological activation of HIFs plays a critical role in cancer progression. In VHL syndrome, affected individuals cary one mutant copy of the gene encoding VHL in their germline. If the other copy of the gene becomes inactivated by mutation in a kidney cel, HIF-1α and HIF- 2α are no longer subject to O2-dependent degradation (which requires VHL) and their dysregulated expression (particularly HIF-2α) drives development of the clear cel type of renal cel carcinoma (RCC) in afected individuals. Beyond this hereditary cancer predisposition syndrome, extensive 1 43011.601_P18269-03clinical and experimental data indicate that both HIF-1 and HIF-2 drive the progression of many cancer types, indicating the need for dual inhibitors of both factors.

[0006] Given their role in disease progression, interest has grown in targeting HIFs for cancer therapy. Thus far, however, only one drug, belzutifan (Welireg™, Merck), which targets HIF-2 only, has been approved by the U.S. Food and Drug Administration for adult patients with VHL disease who require therapy for associated RCC, central nervous system (CNS) hemangioblastomas, or pancreatic neuroendocrine tumors (pNET). Although other smal molecule inhibitors of HIF-1 or HIF-1 and HIF-2 have been identified, there are no known smal molecule agents capable of binding to specific domains of both HIF-1α and HIF- 2α, and inhibiting tumor growth in vivo. The existence of multiple HIF inhibitors binding to distinct molecular sites would be advantageous in the therapeutic response to acquired drug resistance in cases where such resistance is due to mutations that alter the drug binding site. SUMMARY

[0007] In some aspects, the presently disclosed subject mater provides a compound of formula (I): ;

[0010] is a double bond that can be present or absent;

[0011] L1 is a linker selected from –(CH2)t-, -O-CH2-CH2-, and -CH2-CH2-O-; wherein t is an integer selected from 1, 2, and 3;

[0012] A1 and A2 are each independently -CH- or N;

[0013] B1 and B2 can be present or absent and when present are each independently selected from -CR1R2- and -C(C=O)-, wherein R1 and R2 are each independently H or C1-C4 alkyl;

[0014] X1 and X2 are each independently selected from S, O, and SR3, wherein R3 is C1-C4 alkyl;

[0015] Y1 and Y2 are each independently selected from -CH2-, -NR4-, and -O-, wherein R4 is C1- C4 alkyl, or Y1 is -N- when X1 is -SR3 and / or Y2 is -N- when X2 is -SR3.

[0016] Z1 and Z2 are each independently selected from C1-C4 alkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl; and

[0017] stereoisomers and pharmaceuticaly acceptable salts thereof. 2 43011.601_P18269-03

[0018] In certain aspects, L1 is -(CH2)3- and the compound of formula (I) is a compound of formula (Ia): . selected from:O and the compound of formula(Ia-i) and compound of formula (Ia-i) are:of formula (Ia-a) and compound of formula (Ia-i) are:independently selected from substituted or unsubstituted C1-C8 straight chain or branched alkyl, phenyl, pyrimidinyl, imidazoyl, pyrazinyl, and pyridazinyl. 3 43011.601_P18269-03

[0030] In certain aspects, Z1 and Z2 are each independently selected from: ;

[0033] n is an integer selected from 0, 1, 2, 3, 4, and 5;

[0034] m is an integer selected from 0, 1, 2, 3, and 4;

[0035] p is an integer selected from 0, 1, 2 and 3; and

[0036] q is an integer selected from 0, 1, and 2; and

[0037] each R5 is independently selected from halogen, C1-C4 alkyl, hydroxyl, alkoxyl, cyano, - CF3, -NO2, -C(=O)H, -SR6, -SO2R7, -C(=O)-R8, and -C(=O)-O-R9, wherein R6, R7, R8, and R9 are independently C1-C4 alkyl.

[0038] In particular aspects, the compound of formula (I) is selected from a compound listed in Table 10.

[0039] In more particular aspects, the compound of formula (I) is selected from: ;compound of formula (I): 4 43011.601_P18269-03);

[0046] L2 is a linker which can be present or absent and when present is selected from: ; aninteger selected from 0, 1, 2, and 3, each X3 can be present or absent and when present is -O-, and R13 is selected from H, C1-C4 alkyl, and -NR14R15, wherein R14 and R15 are each H or C1-C4 alkyl;

[0049] R10 is selected from a substituted or unsubstituted cycloalkyl or heterocycloalkyl, straight chain or branched C1-C4 alkyl, C1-C4 alkoxyl, -C(=O)-O-R16, and -CH2-C(=O)-O-R17, wherein R16 and R17 are each independently C1-C4 alkyl;

[0050] R11 is selected from H, straight chain or branched C1-C4 alkyl, -NR18R19, wherein R18 and R19 are each H or C1-C4 alkyl;

[0051] R12 is selected from H and halogen; or

[0052] R10 and R12 together form a 5- to 6- membered cycloalkyl or heterocycloalkyl ring;

[0053] Z3 is selected from C1-C4 alkyl, aryl, and heteroaryl, each of which can be substituted or unsubstituted; and

[0054] stereoisomers and pharmaceuticaly acceptable salts thereo.

[0055] In certain aspects, ring C is selected from: .(I) is selected from:5 43011.601_P18269-03

[0059] (Ib).

[0060] 10 is a substituted or unsubstituted cycloalkyl or heterocycloalkyl selected from: ;

[0063] R20 is selected from H, C1-C4 alkyl, hydroxyl, and -O-C(=O)-R24, wherein R24 is C1-C4 alkyl;

[0064] R21 is H or C1-C4 alkyl;

[0065] R22 is selected from H, C1-C4 alkyl, and phenyl; or

[0066] R20 and R21 together form a 1,3-dioxolane ring structure.

[0067] In certain aspects, Z3 is selected from:;6 43011.601_P18269-03

[0069] wherein:

[0070] n3 is an integer selected from 0, 1, 2, 3, 4, and 5;

[0071] m1 is an integer selected from 0, 1, 2, 3, and 4;

[0072] p1 is an integer selected from 0, 1, 2 and 3; and

[0073] q1 is an integer selected from 0, 1, and 2; and

[0074] each R25 is independently selected from halogen, C1-C4 alkyl, hydroxyl, alkoxyl, cyano, - CF3, -NO2, -C(=O)H, -SR26, -SO2R27, -C(=O)-R28, and -C(=O)-O-R29, wherein R26, R27, R28, and R29 are independently C1-C4 alkyl.

[0075] In particular aspects, the compound of formula (I) is selected from a compound listed in Table 11.

[0076] In more particular aspects, the compound of formula (I) is selected from: .a compound of formula (I) or formula (I) and an pharmaceuticaly acceptable carrier. In particular aspects, formulation comprises a liposomal composition, niosomes, nanoemulsions, nanosuspensions, miceles, including nanomiceles, hydrogels, polymeric nanoparticles, including PLGA nanoparticles and other biocompatible polymeric nanoparticles, solid lipid nanoparticles, nanostructured lipid carriers, nanocrystals, dendrimers, cubosomes, olaminosomes, bilosome, extracelular vesicles (exosomes), and smart nano-micro platforms.

[0080] In some aspects, the formulation comprises a compound of formula (I) or formula (I) incorporated a microparticle or microsphere comprising one or more biodegradable polymers including, but not limited to, a poly(lactic-co-glycolic)acid (PLGA), a poly(ε-caprolactone) (PCL), a poly(lactic acid) (PLA), a poly(glycolic acid) (PGA), a polyester, a poly(orthoester), a poly(phosphazine), a poly(phosphate ester), a poly(ε-caprolactone-co-ethyl ethylene phosphate) 7 43011.601_P18269-03(PCLEEP), a polyvinyl alcohol (PVA), a poly(β-amino ester), a poly(acrylic acid) (PAA), a poly- 3-hydroxybutyrate (P3HB), a poly(hydroxybutyrate-co-hydroxyvalerate), a polyethylene glycol (PEG), and combinations thereof.

[0081] In other aspects, the presently disclosed subject mater provides a method for treating a disease, disorder, or condition associated with one or more hypoxia inducible factors (HIFs) in a subject in need of treatment thereof, the method comprising administering to the subject a therapeuticaly efective amount of a compound of formula (I) or formula (I) or a formulation thereof.

[0082] In certain aspects, the disease, disorder, or condition is a cancer. In particular aspects, the cancer is selected from breast cancer, colorectal cancer, lung cancer, melanoma, pancreatic cancer, prostate cancer, and head and neck squamous cel carcinoma (HNSCC). In certain aspects, administration of the compound of formula (I) or formula (I) inhibits or blocks growth and / or vascularization of a tumor associated with breast cancer, colorectal cancer, lung cancer, melanoma, pancreatic cancer, or prostate cancer.

[0083] In some aspects, the method further comprises administering one or more additional therapeutic agents. In certain aspects, the one or more additional therapeutic agents include an anti- cancer agent. In particular aspects, the anti-cancer agent is selected from an angiogenesis inhibitor, an angiopoietin 2 inhibitor, a CD73 inhibitor, a cyclin dependent kinase inhibitor, an MAP kinase inhibitor, a mTOR inhibitor, a phosphatidylinositol 3-kinase inhibitor, a proteasome inhibitor, a protein phosphatase 2A activator, a serine / threonine kinase inhibitor, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a VEGF-A inhibitor, and a cytotoxic chemotherapy, including, but not limited to, 5-fluorouracil, adriamycin, carboplatin, cisplatin, doxorubicin, gemcitabine, idarubicin, and paclitaxel.

[0084] In other aspects, the method further comprises administering the compound of formula (I) or formula (I) in combination with an immunotherapy. In certain aspects, the immunotherapy comprises immune checkpoint blockade (ICB) immunotherapy. In particular aspects, the ICB immunotherapy includes anti-CTLA-4, anti-PD-1, or anti-PD-L1 immunotherapy. In more particular aspects, administering the compound of formula (I) or formula (I) in combination with the immunotherapy alters an immune cel microenvironment of a tumor associated with breast cancer, colorectal cancer, lung cancer, melanoma, pancreatic cancer, prostate cancer, or head and neck squamous cel carcinoma (HNSCC). In yet more particular aspects, administering the 8 43011.601_P18269-03compound of formula (I) or formula (I) in combination with an immunotherapy reduces a risk of an adverse event associated with the immunotherapy.

[0085] In certain aspects, the disease, disorder, or condition is associated with ocular neovascularization. In particular aspects, the disease, disorder, or condition associated with ocular neovascularization is selected from diabetic macular edema, diabetic retinopathy, retinal vein occlusion, sickle cel retinopathy, retinopathy of prematurity, Norrie’s disease, Coat’s disease, corneal neovascularization, and age-related macular degeneration. In certain aspects, administration of the compound of formula (I) decreases the expression of VEGF and ANGPTL4.

[0086] In some aspects, the method further comprises administering one or more additional therapeutic agents. In certain aspects, the one or more additional therapeutic agents include an agent for treating a disease, disorder, or condition associated with ocular neovascularization. In particular aspects, the one or more additional therapeutic agents for treating a disease, disorder, or condition associated with ocular neovascularization include an anti-VEGF agent. In more particular aspects, the anti-VEGF agent is aflibercept, bevacizumab, faricimab, or ranubizumab.

[0087] In certain aspects, the compound of formula (I) or formula (I) is administered prophylacticaly to prevent or reduce an incidence, recurrence, or progression of the disease, disorder, or condition.

[0088] In certain aspects, the compound of formula(I) or formula (I): (a) directly binds the bHLH domain or the PAS-B subdomain of HIF-1α and HIF-2α; (b) disrupts dimerization with HIF-1β; (c) induces degradation of HIF-1α and HIF-2α; and (d) inhibits HIF target gene expression in one or more cels of the subject.

[0089] Certain aspects of the presently disclosed subject mater having been stated hereinabove, which are addressed in whole or in part by the presently disclosed subject mater, other aspects wil become evident as the description proceeds when taken in connection with the accompanying Examples and Figures as best described herein below. BRIEF DESCRIPTION OF THE FIGURES

[0090] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings wil be provided by the Ofice upon request and payment of the necessary fee. 9 43011.601_P18269-03

[0091] Having thus described the presently disclosed subject mater in general terms, reference wil now be made to the accompanying Figures, which are not necessarily drawn to scale, and wherein:

[0092] FIG.1A, FIG.1B, FIG.1C, FIG.1D, FIG.1E, FIG.1F, and FIG.1G ilustrate the discovery and characterization of dual HIF-1 / 2 inhibitors. (FIG.1A) Pharmacophore features at sites 1, 2, and 3 are overlaid on HIF-2α (grey) and HIF-1β (pink) and shown as red (H-bond acceptor), blue (H-bond donor) and cyan (hydrophobic) spheres. (FIG.1B) Occupancy of site 1 and site 3 by SS1.21 and SS3.2, respectively, is shown. (FIG.1C) HIF-α subunit domain structure is shown. (FIG.1D) Chemical structures of dual HIF-1 / 2 inhibitors are shown. (FIG.1E and FIG.1F) Expression of CA9 (FIG.1E) and EPO (FIG.1F) mRNA was analyzed in Hep3B cels incubated for 24 hours at 20% or 1% O2 with vehicle, or at 1% O2 with the indicated HIF inhibitor at the indicated concentration (μM). Results are presented as mean ± SD (n = 3). #P < 0.05 vs 1% O2 (vehicle); ns, no significant diference vs 1% O2 (vehicle); two-way ANOVA with Dunnet’s multiple comparisons post-test. FIG.1G shows the chemical structures of representative compounds tested.

[0093] FIG.2A and FIG.2B show expression of RPL13A and HIF mRNAs in Hep3B cels. FIG. 2A and FIG.2B, Hep3B cels were treated with 0 to 10 μM of the indicated dual HIF inhibitor (SS1.21, 1.21S9N, SS3.2, or 3.2.16) or HIF-2-selective inhibitor (PT2385 or PT2977) for 24 hours, and mRNA expression was determined by RT-qPCR assays. Data are presented as mean + SD (n = 3). ns, no significant diference vs vehicle-treated cels; two-way ANOVA with Dunnet’s multiple comparisons post-test.

[0094] FIG.3A, FIG.3B, FIG.3C, FIG.3D. FIG.3E, FIG.3F, FIG.3G, FIG.3H, FIG.3I, FIG. 3J, FIG.3K, FIG.3L, FIG.3M, FIG.3N, FIG.3O, and FIG.3P . SS1.21 and SS3.2 trigger degradation of HIF-1α and HIF-2α to inhibit HIF target gene expression. (FIG.3A and FIG.3B) Hep3B cels were treated with 10-μM SS1.21 (FIG.3A) or SS3.2 (FIG.3B) at 20% or 1% O2 for 24 hours and immunoblot assays were performed. (FIG.3C) Cels transfected with vector encoding HIF-1α DM (P402A / P564A) were treated with vehicle, 10-μM SS1.21 (top), or SS3.2 (botom) for 6 hours. (FIG.3D- FIG.3G) Cels were treated with vehicle or 1 mM DMOG and vehicle or either 10-μM SS1.21 (FIG.3D and FIG.3E) or SS3.2 (FIG.3F and FIG.3G) for 6 hours (FIG.3D and FIG.3F) or 24 hours (FIG.3E and FIG.3G). (FIG.3H and FIG.3I) Cels were treated with 5-μM SS1.21 (FIG.3H) or SS3.2 (FIG.3I) in the presence of vehicle (V), 10-μM MG132 or 10- 10 43011.601_P18269-03nM bafilomycin (Baf) at 20% or 1% O2 for 6 hours. (FIG.3J and FIG.3K) Cels were treated with 5-μM SS1.21 (FIG.3J) or SS3.2 (FIG.3K) with V or 500-nM TAK243 for 6 hours. (FIG.3L and FIG.3M) Cels were treated with 5-μM SS1.21 (FIG.3L) or SS3.2 (FIG.3M) with V or MG132 and immunoprecipitation (IP) was performed using anti-HIF-1β antibody. Inputs and immunoprecipitates were subjected to immunoblot assays. (FIG.3N and FIG.3O) Ful length (FL) HIF-1α and deletion mutants (ΔbHLH and ΔPAS; FIG.3N) were transiently expressed in Hep3B cels, which were treated with 5-μM SS1.21 (FIG.3O, left) or SS3.2 (FIG.3O, right). (FIG.3P) IC50 values are shown for inhibition of target gene expression in cancer cels treated with indicated HIF inhibitor. See FIG.2C-FIG.2D for additional loading controls for panels A, B, L and M.

[0095] FIG.4A, FIG.4B, FIG.4C, FIG.4D. FIG.4E, FIG.4F, FIG.4G, FIG.4H, and FIG.4I. SS1.21 or 3.2.16 administration inhibits colorectal cancer growth. (FIG.4A and FIG.4B) Nude mice bearing 150-mm3 HCT116 xenografts were treated with vehicle or SS1.21 and tumor growth was monitored (mean + SD, n = 5); *P < 0.05 by two-way ANOVA (FIG.4A). Tumors were harvested 4 hours after the last dose and immunoblot assays were performed (FIG.4B). (FIG.4C) Mice were injected subcutaneously with HCT116 cels and, when a tumor was palpable, were treated with vehicle, oxaliplatin (Oxa), SS1.21, or Oxa + SS1.21 and tumor volumes were determined (mean + SD, n = 5); *P < 0.05 by two-way ANOVA. (FIG.4D) Balb / c mice bearing 100-mm3 CT26 tumors were treated with vehicle or SS1.21 and tumor volumes were determined (mean + SD, n = 5); *P < 0.05 by two-way ANOVA with Bonferoni post-test. (FIG.4E) Tumors were harvested 4 hours after the last dose and immunoblot assays were performed. (FIG.4F) Tumor sections were analyzed by immunohistochemistry using anti-CD31 antibody. (FIG.4G) C57BL / 6 mice were injected with MC38 cels and treated with vehicle, α-CTLA-4 (200 μg Q3D), 3.2.16 (40 mg / kg BID) or both. (FIG.4H) Kaplan-Meier analysis of mouse survival is shown. (FIG.4I) Mean and individual tumor growth are presented. ****P < 0.0001 vs α-CTLA-4 by two- way ANOVA with Tukey’s post-test.

[0096] FIG.5A, FIG.5B, FIG.5C, FIG.5D, FIG.5E, and FIG.5F are measurements of body weight, tumor weight and mean tumor volume of tumor-bearing mice. FIG.5A to FIG.5D, Mice bearing HCT116 (FIG.5A- FIG.5C) or CT26 (FIG.5D) tumors were treated with vehicle, SS1.21, oxaliplatin (Oxa), or SS1.21 + Oxa (Combination) and body weights and tumor weight were measured every 1-4 days. Data are presented as mean + SD (n = 5). FIG.5E, Quantification of CD31+ vessel area (shown in FIG.4F). FIG.5F, Mean tumor volume of MC38 mouse model 11 43011.601_P18269-03(presented in FIG.4I-FIG.4L) for up to 21 days. Results are presented as mean ± SD (n = 3). *** P < 0.05 vs 1% O2 (vehicle); two-way ANOVA with Dunnet’s multiple comparisons post-test.

[0097] FIG.6A, FIG.6B, and FIG.6C demonstrate the efect of SS1.21 on growth of head and neck squamous cel carcinoma (HNSCC). FIG.6A and FIG.6B, Human FaDu (FIG.6A) and mouse SCCVI (FIG. S4B) HNSCC cels were injected into nude and syngeneic C3H mice, respectively, and when tumors reached 150 mm3, the mice were treated with vehicle or SS1.21 (10 or 20 mg / kg twice daily × 5 days) by intraperitoneal injection. Tumor volumes were determined and are presented as mean + SD (n = 5). *P < 0.05, **P < 0.01 by two-way ANOVA with Tukey’s post test; ns, no significant diference. FIG.6C, Representative hematoxylin and eosin staining of FaDu tumor sections from vehicle- and SS1.21-treated mice showing tumor margins. Tumors from vehicle-treated mice were dificult to resect due to extensive infiltration of surounding skeletal muscle by invasive FaDu cels, whereas tumors from mice treated with SS1.21 were easily resected with clean margins (botom of fields). Scale bars represent 500 µm.

[0098] FIG.7A, FIG.7B, FIG.7C, and FIG.7D show treatment of breast cancer with HIF inhibitors. FIG.7A and FIG.7B, BT-474 human breast cancer cels were injected into the mammary fat pad (MFP) of nude mice and when tumors reached a volume of 150 mm3, the mice were treated with SS3.2 (10 mg / kg BID IP × 7 days) and tumor volume was determined daily (FIG.7A). Data shown are mean + SD (n = 5). *P < 0.05 by two-way ANOVA. Tumors were resected 4 hours after the last dose and lysates were analyzed for expression of HIF-1α, HIF-1β, and β-actin by immunoblot assays (FIG.7B). FIG.7C, Tumor-free mice from the EMT6 study shown in FIG.10A-FIG.10H were rechalenged, and naïve mice were chalenged, with an MFP injection of EMT6 cels and tumor volumes were determined. Data are presented as mean + SD (n = 4). FIG.7D, EMT6 cels were injected into the MFP of syngeneic Balb / c mice and when the tumors reached a volume of 150 mm3, the mice were treated with vehicle or with 1.21S9N or 3.2.16 at 40 mg / kg BID IP × 5 days and tumor volume was determined daily. Data are presented as mean + SD (n = 5), *P < 0.05 by two-way ANOVA with Tukey’s post-test. Tumors were harvested 4 hours after the last dose and were used to generate the data presented in FIG.10I-FIG. 10J.

[0099] FIG.8A, FIG.8B, and FIG.8C show treatment of breast cancer with HIF inhibitors. FIG. 8A to FIG.8C, Mean tumor volumes (for the E0771 study shown in FIG.9A-FIG.9F); data are presented as mean + SD (n = 5); *P < 0.05 by two-way ANOVA with Tukey’s post-test (FIG.8A). 12 43011.601_P18269-03E0771 mean tumor weights at the time of termination, 4 hours after the last dose, is shown (FIG. 8B). Mice that remained tumor-free after combination therapy were rechalenged with a second injection of E0771 cels, as wel as naïve mice, and tumor volumes are presented as mean + SD (FIG.8C).

[0100] FIG.9A, FIG.9B, FIG.9C, FIG.9D. FIG.9E, FIG.9F, FIG.9G, FIG.9H, FIG.9I, FIG. 9J, FIG.9K, and FIG.9L. Efect of treatment with 3.2.16 in E0771 BrCa model. (FIG.9A) C57BL / 6 mice received a mammary fat pad injection of E0771 cels and were treated with vehicle or IgG2a isotype-control antibody; α-CTLA-4; 3.2.16; or combination. (FIG.9B, FIG.9C, FIG. 9E, FIG.9F) For each treatment group, individual tumor growth curves are presented. The number of mice in each group that achieved a complete response (CR) on day 27 is indicated. (FIG.9D) Tumors from mice treated with vehicle or 3.2.16 were analyzed for mRNA expression, shown as mean + SD (n = 5); *P < 0.05 by Student’s t test. (FIG.6G-FIG.6L) Mice with 150-mm3 E0771 tumors were treated with vehicle, α-CTLA-4, 3.2.16, or α-CTLA-4 + 3.2.16 (combination) and analyzed by flow cytometry. The percentage of CD45+ tumor-infiltrating leukocytes (TILs; G), CD45+ CD3+ T cels (FIG.9H), CD45+ CD3- NK1.1+ IFNγ+ NK cels (FIG.9I), CD45+ CD11b+ F4 / 80+ tumor-associated macrophages (TAMs; FIG.9J), CD45+ CD11b+ Ly6G+ myeloid-derived suppressor cels (MDSCs; FIG.9K), and CD45- CD3- PDL1+ tumor cels (FIG.9L) were determined. Data are presented as mean + SD (n = 5). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by one-way ANOVA with Dunnet’s post-test.

[0101] FIG.10A, FIG.10B, FIG.10C, FIG.10D. FIG.10E, FIG.10F, FIG.10G, FIG.10H, FIG. 10I, FIG.10J, and FIG.10K. Efect of 1.21S9N or 3.2.16 in breast cancer model. (FIG.10A) EMT6 cels were injected into the mammary fat pad of Balb / c mice, which were treated with: vehicle or IgG2a isotype-control antibody; α-CTLA-4; HIFi (1.21S9N or 3.2.16); or α-CTLA-4 + HIFi. (FIG.10B) Kaplan-Meier analysis of mouse survival is shown. (FIG.10C-FIG.10H) For each treatment group, individual tumor growth curves are shown. The number of mice in each group that achieved a durable complete response (CR) on day 100 is indicated. (FIG.10I and FIG. 10J) Mice with 150-mm3 EMT6 tumors were treated with vehicle or 1.21S9N or 3.2.16 (40 mg / kg BID) for 5 days, tumors were harvested 4 hours after the last dose, and mRNA (FIG.10I) and protein (FIG.10J) expression were analyzed by RT-qPCR (mean + SD, n = 5; *P < 0.05 by one- way ANOVA with Bonferoni post-test) and immunoblot assays, respectively. (FIG.10K- FIG. 10L) Tumor growth curves for individual mice (FIG.10L) and survival (FIG.10K) of MMTV- 13 43011.601_P18269-03PyMT mic FIG.10E treated with either vehicle or 1.21S9N (60 mg / kg BID OG) starting on day 53 of life. Note that the five mice (designated m1-5 in the smal graph at botom right) treated with 1.21S9N did not develop any mammary tumors during the treatment period.

[0102] FIG.11A, FIG.11B, FIG.11C, FIG.11D, FIG.11E, and FIG.11F. Efect of treatment with 1.21S9N or 3.2.16 in melanoma and prostate cancer models. (FIG.11A) C57BL / 6 mice were injected subcutaneously with B16F10 melanoma cels and treated with vehicle, HIFi (1.21S9N or 3.2.16), α-CTLA-4, or HIFi + α-CTLA-4. (FIG.11B) Mean tumor volume is shown. ****P < 0.0001 vs α-CTLA-4 by two-way ANOVA with Tukey’s post-test. (FIG.11C) Kaplan-Meier analysis of mouse survival is shown. (FIG.11D) C57BL / 6 mice were injected with DX1 prostate cancer cels and treated with vehicle, HIFi, α-CTLA-4, or HIFi + α-CTLA-4. (FIG.11E) Mean tumor volume is shown. *P < 0.05; **P < 0.01 vs α-CTLA-4 by two-way ANOVA with Tukey’s post-test. (FIG.11F) Kaplan-Meier analysis of relapse-free survival is shown.

[0103] FIG.12A, FIG.12B, FIG.12C, FIG.12D, FIG.12E, and FIG.12F are tumor growth curves for C57BL / 6 mice injected subcutaneously with B16F10 melanoma cels and treated with vehicle (FIG.12A), α-CTLA-4 (FIG.12B), HIFi (1.21S9N or 3.2.16) (FIG.12C and FIG.12D), α-CTLA- 4, or HIFi + α-CTLA-4 (FIG.12E and FIG.12F). Individual tumor growth curves are presented.

[0104] FIG.13A, FIG.13B, FIG.13C, FIG.13D, FIG.13E, FIG.13F, FIG.13G, and FIG.13H show treatment of mouse prostate cancer model with HIF inhibitors. FIG.13A to FIG.13H, C57BL / 6 mice were injected with DX1 prostate cancer cels and treated with vehicle, HIFi, α- CTLA-4, or HIFi + α-CTLA-4. Individual tumor growth curves are shown (FIG.13A- FIG.13F). FIG.13G and FIG.13H, DX1 cels were injected subcutaneously in syngeneic C57BL6 mice and when the tumors reached a volume of 150 mm3, the mice were treated with vehicle or with 1.21S9N or 3.2.16 at 40 mg / kg BID IP × 5 days and tumor volume was determined daily; data are presented as mean + SD (n = 5), *P < 0.05, **P < 0.01 by two-way ANOVA with Tukey’s post test (FIG.13G). Tumors were harvested 4 hours after the last dose, RNA was isolated, and analyzed by RT-qPCR; data are presented as mean + SD (n = 5); *P < 0.05 by one-way ANOVA with Bonferoni post-test (FIG.13H).

[0105] FIG.14a, FIG.14b, FIG.14c, and FIG.14d demonstrate identification of ligand binding sites in HIF-2α using SILCS. FIG.14a. SILCS Fragmaps of HIF-2α. FragMap types are shown as grids for the aliphatic (green, -1.2 kcal / mol), aromatic (purple, -1.2 kcal / mol), hydrogen-bond donor (blue, -0.9 kcal / mol) and hydrogen-bond acceptor (red, -0.9 kcal / mol), positively charged 14 43011.601_P18269-03(cyan, -1.5 kcal / mol) and negatively (orange, -1.5 kcal / mol) charged groups. FIG.14b. Exclusion map (solid bronze surface) overlaid with the FragMaps. FIG.14c. Pharmacophore features in sites 1, 2, and 3 (marked by red circles) overlaid on HIF-2α (grey cartoon) and HIF-1β (pink cartoon). Pharmacophore features (spheres) are shown as green (apolar), red (H-bond acceptor), blue (H- bond donor) and cyan (cationic). The final pharmacophore hypotheses used for virtual screening contained subsets of these features (refer to Table 2 and Table 3). FIG.14d. Representative ligands at sites 1, 2, and 3 along with SILCS Fragmaps.

[0106] FIG.15A and FIG.15B are immunoblot assays of blots with actin loading controls. These data are an expansion of data presented in FIG.3A, FIG.3B, FIG.3L and FIG.3M, where only a single representative actin loading control is shown. Here the β-actin loading control for each blot is shown.

[0107] FIG.16A and FIG.16B. Complete responses and survival analysis. (FIG.16A) The aggregate complete responses in mice with transplanted tumors treated with immune checkpoint blockade (ICB) or with ICB + HIF inhibitor (ICB + HIFi) are presented. (FIG.16B) Aggregate Kaplan-Meier survival curves for mice treated with vehicle, ICB, HIFi, or ICB + HIFi. ****P < 0.0001 vs ICB by log-rank test.

[0108] FIG.17A, FIG.17B, FIG.17C, FIG.17D. FIG.17E, and FIG.17F, Eficacy and safety of dual HIF-1 / 2 inhibition compared to selective HIF-2α inhibition in mice. (FIG.17A) Balb / c mice bearing CT26 tumors were treated by oral gavage (OG) with either vehicle, PT2385 (60 mg / kg QD) or 1.21S9N (60 mg / kg QD) or α-PD-1 (200 μg Q3D, IP) in combination with either PT2385 (60 mg / kg QD) or 1.21S9N (60 mg / kg QD) and tumor volumes were determined (mean + SD, n = 5). ****P < 0.0001 for 1.21S9N vs PT2385; ####P < 0.0001 for 1.21S9N + α-PD-1 vs PT2385 + α-PD-1 by two-way ANOVA with Tukey’s post-test. (FIG.17B) Kaplan-Meier analysis of survival is shown. (FIG.17C and FIG.17D) Tumors were harvested 4 hours after the last dose for analysis of mRNA expression (FIG.17C) and immunohistochemistry using anti-CD31 antibody (FIG.17D). Results are presented as mean ± SD (n = 5). *P < 0.05; **P < 0.01 vs vehicle; one- way ANOVA with Bonferoni post-test. (FIG.17E and FIG.17F) Breathing was monitored pre- and post-treatment with vehicle, 1.21S9N or PT2385 (30 mg / kg BID OG × 3 doses) by whole body plethysmography and representative ventilatory tracings are shown (FIG.17E). For each mouse, the apnea index (events per hour) is ploted as mean ± SEM for each treatment group (n = 5 mice per group); ***P < 0.001 vs vehicle by two-way ANOVA with Holm-Sidak post-test (FIG.17F). 15 43011.601_P18269-03

[0109] FIG.18A, FIG.18B, FIG.18C, FIG.18D, FIG.18E, FIG.18F, FIG.18G are: FIG.18A to FIG.18C, Nude mice were injected subcutaneously with HCT116 colorectal carcinoma cels and oraly treated with vehicle, 1.21S9N, or 3.2.16. Body weight (FIG.18A) and mean tumor volume (FIG.18B) are presented as mean ± SD (n = 5); (P < 0.05, 2-way ANOVA with Bonferoni post- test). Tumor tissues were colected at the indicated time points and analyzed for HIF-1α protein expression (FIG.18C). FIG.18D, CT26 cels were incubated for 24 hours at 20% or 1% O2 (with vehicle) or at 1% O2 with the indicated HIF inhibitor at the indicated concentration (mM). Ca9 and Adm mRNA levels were determined by RT-qPCR. Results are presented as mean ± SD (n = 3). *** P < 0.05 vs 1% O2 (vehicle); two-ay ANOVA with Dunnet’s multiple comparisons post-test. FIG.18E to FIG.18G, Tumors from mice treated with vehicle (FIG. S9E), 1.21S9N (FIG. S9F), or PT2385 (FIG.18G) from the experiment shown in FIG.16A were analyzed by immunohistochemistry using anti-CD31 antibody. Fields at 10×, 20×, and 40× magnification are shown.

[0110] FIG.19A, FIG.19B, and FIG.19C show that compounds 3.2n and 3.2.16n demonstrate strong anti-tumor activity in a model of HER2+ breast cancer. FIG.19A shows the tumor volume (mm3) vs. days post-treatment for vehicle, compound 3.2n (20 mg kg-1 IP BID), and compound 3.2.16n (40 mg kg-1 IP BID). FIG.19B shows photographs of tumors removed from the mammary fat pad of immunodeficient mice after implantation of human HER2+ breast cancer cels and treatment with the compounds at the indicated doses for 7 days once the tumors reached a volume of 150 mm3. FIG.19C shows the tumor weight (g) vs. days post-treatment for vehicle, compound 3.2n (20 mg kg-1 IP BID), and compound 3.2.16n (40 mg kg-1 IP BID).

[0111] FIG.20A, FIG.20B, FIG.20C, and FIG.20D demonstrate that compound 3.2n was active in the HCT116 human colorectal cancer xenograft model. FIG.20A shows the tumor volume (mm3) vs. days post-treatment for vehicle, compound 1.21S9N (40 mg kg-1 IP BID), and compound 3.2n (20 mg kg-1 IP BID). FIG.20B shows the tumor volume (mm3) vs. days post- treatment for vehicle. FIG.20C shows the tumor volume (mm3) vs. days post-treatment for compound 1.21S9N (40 mg kg-1 IP BID), FIG.20D shows the tumor volume (mm3) vs. days post- treatment for compound 3.2n (20 mg kg-1 IP BID).

[0112] FIG.21A, FIG.21B, FIG.21C, FIG.21D, FIG.21E, FIG.21F, FIG.21G, and FIG.21H demonstrate that compound 3.2n was active in the orthotopic EMT6 mouse triple-negative breast cancer model. FIG.21A shows the tumor volume (mm3) vs. days post-treatment for vehicle, 16 43011.601_P18269-03compound 1.21S9N (40 mg kg-1 IP BID), compound 3.2n (20 mg kg-1 IP BID), compound 3.2.16 (40 mg kg-1 IP BID), and compound 3.2.67n (40 mg kg-1 IP BID). FIG.21B shows the tumor volume (mm3) vs. days post-treatment for vehicle. FIG.21C shows the tumor volume (mm3) vs. days post-treatment for compound 1.21S9N (40 mg kg-1 IP BID). FIG.21D shows the tumor volume (mm3) vs. days post-treatment for compound 3.2n (20 mg kg-1 IP BID). FIG.21E shows the tumor volume (mm3) vs. days post-treatment for compound 3.2.16 (40 mg kg-1 IP BID). FIG. 21F shows the tumor volume (mm3) vs. days post-treatment for compound 3.2.67n (40 mg kg-1 IP BID). FIG.21G shows an immunoblot assay for cels treated with vehicle, compound 1.21S9N, compound 3.2n, compound 3.2.16, and compound 3.2.67n. FIG.21H shows tumor volume at termination for vehicle, compound 1.21S9N, compound 3.2n, compound 3.2.16, and compound 3.2.67n.

[0113] FIG.22A and FIG.22B show dose-responses and derived IC50 values for compounds SS1.21 and derivative 1.21S9N; and SS3.2 and derivatives 3.2n, 3.2.16, and 3.2.16n, as wel as the HIF-2 selective inhibitors from Peleton, PT2385 and PT2399, on the expression of the HIF-1 target gene CA9 and the HIF-2 target gene EPO.

[0114] FIG.23 shows a summary of IC50 values for compounds SS1.21 and derivative 1.21S9N; and SS3.2 and derivatives 3.2n, 3.2.16, and 3.2.16n.

[0115] FIG.24A, FIG.24B, and FIG.24C show that treatment with 3.2.67n (40 mg / kg BID) monotherapy decreased EMT6 tumor growth but the efect was not statisticaly significant (FIG. 24A). Tumors from mice treated with 1.21S9N, 3.2n, 3.2.16, or 3.2.67n showed a marked loss of HIF-1a protein expression (FIG.24B). Treatment of mice bearing HCC1954 human HER2+ orthotopic breast tumors with 3.2n or 3.2.16n monotherapy for 7 days significantly inhibited tumor growth (FIG.24C).

[0116] FIG.25A, FIG.25B, FIG.25C, FIG.25D, FIG.25E, and FIG.25F demonstrate treatment of mice injected with Lewis lung carcinoma (LLC) cels. C57BL / 6 mice were injected subcutaneously with Lewis lung carcinoma (LLC) cels and treated with vehicle, 1.21S9N, α-PD- 1, or 1.21S9N + α-PD-1 as indicated (FIG.25A). Mouse survival (FIG.25B) and tumor growth (FIG.25C-FIG.25F) are presented (SD = stable disease).

[0117] FIG.26A, FIG.26B, FIG.26C, FIG.26D, FIG.26E, FIG.26F, FIG.26G, FIG.26H, and FIG.26I demonstrate the efect of 1.21S9N and 3.2.16 in pancreatic and prostate cancer models. (FIG.26A to FIG.26H) C57BL / 6 mice were injected subcutaneously with KPC pancreatic 17 43011.601_P18269-03adenocarcinoma cels and treated with vehicle, immune checkpoint blockade (ICB; α-CTLA-4 or α-PD-1), 3.2.16, or ICB + 3.2.16 (FIG.26A) and tumor growth is presented as mean ± SD (n = 5); (P < 0.05, 2-way ANOVA with Bonferoni post-test) (FIG.26B-FIG.26G). CD31 immunohistochemistry was performed on tumor sections from vehicle- or 3.2.16-treated mice (FIG.26H). (FIG.26I) Male C57BL / 6 mice were injected with DX1 prostate cancer cels and treated with vehicle, HIFi (1.21S9N or 3.2.16), α-CTLA-4, or HIFi + α-CTLA-4 (FIG.26I).

[0118] FIG.27A and FIG.27B show treatment of DX1 prostate cancer with HIF inhibitors. (FIG. 27A) DX1 cels were exposed to 20% or 1% O2 in the presence of vehicle, or 1% O2 in the presence of the indicated concentration (µM) of 1.21S9N or 3.2.16, for 24 hours and B7-H3 mRNA expression was analyzed by RT-qPCR. Data are presented as mean + SD (n = 3) (FIG.27B) Mice bearing 100-mm3 DX1 tumors were treated with vehicle or 1.21S9N or 3.2.16 (40 mg / kg BID × 5 days). Tumor volume was determined and data are shown as mean ± SD (n = 5). *P < 0.05 by two- way ANOVA with Tukey’s post test. RNA extracted from these tumors was analyzed in FIG.13H.

[0119] FIG.28A, FIG.28B, FIG.28C, FIG.28D, FIG.28E, and FIG.28F demonstrate the effect of 3.2.16 on the immune cel microenvironment in mouse breast cancer. Mice with 150-mm3 E0771 tumors were treated with vehicle, α-CTLA-4, 3.2.16, or α-CTLA-4 + 3.2.16 (combination) and analyzed by flow cytometry. The percentage of CD45+ tumor-infiltrating leukocytes (TILs; FIG.28A), CD45+ CD3+ T cels (FIG.28B), CD45+ CD3- NK1.1+ IFNg+ NK cels (FIG.28C), CD45+ CD11b+ F4 / 80+ tumor-associated macrophages (TAMs; FIG.28D), CD45+ CD11b+ Ly6G+ myeloid-derived suppressor cels (MDSCs; FIG.28E), and CD45- CD3- PDL1+ tumor cels (FIG. 28F) were determined. Data are presented as mean ± SD (n = 5 in FIG.28B and FIG.28F; n = 10 in al others). *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA with Dunnet’s post-test.

[0120] FIG.29A and FIG.29B show the choroidal neovascularization area (CNV) (mean + standard deviation) calculated for each laser injury site to the choroid of mice folowed by treatment with vehicle control (DMSO) (VE), 32-134D, 3.2.16 (70 ng) (P < 0.01), and 3.2.16 (140 ng) (P < 0.001). FIG.29A is a schematic showing the treatment schedule. FIG.29B shows the dose dependent efect of HIF inhibitor compound (3.2.16) on the CNV lesion size in C57BL / 6 mice. Mice received an intravitreal injection of 70 ng or 140 ng of 3.2.16 given 3 days after laser treatment on day 0. Eyes were enucleated on day 7 (D7), stained with fluorophore-conjugated isolectin B4 and analyzed for lesion size. Data are shown as mean ± SD; n = 4 mice per condition. 18 43011.601_P18269-03Statistical analyses are performed by 2-tailed Student’s t test. * P > 0.05, ** P > 0.01, *** P > 0.001.

[0121] DETAILED DESCRIPTION

[0122] The presently disclosed subject mater now wil be described more fuly hereinafter with reference to the accompanying Figures, in which some, but not al embodiments of the inventions are shown. Like numbers refer to like elements throughout. The presently disclosed subject mater may be embodied in many diferent forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure wil satisfy applicable legal requirements. Therefore, it is to be understood that the presently disclosed subject mater is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims.

[0123] HYPOXIA INDUCIBLE FACTOR (HIF)-1 / 2 DUAL INHIBITORS

[0124] The presently disclosed subject mater provides smal molecule inhibitors of HIF-1α and HIF-2α, which target specific domains of HIF-1α and HIF-2α and induce the degradation of these proteins. As provided in more detail herein below, the presently disclosed compounds block the expression of HIF-regulated genes in human and mouse melanoma cels, as wel as breast, colorectal, cervical, lung, ovarian, pancreatic, prostate, and head and neck squamous cel carcinoma (HNSCC) cancer cels. When administered to tumor-bearing mice, the compounds inhibited the growth of breast, colorectal, lung, pancreatic and prostate cancer, and HNSCC. Further, when administered in combination with immunotherapy, these compounds caused tumor eradication in mouse models of melanoma, breast, colorectal, prostate, and pancreatic cancer.

[0125] Initialy, the Site Identification by Ligand Competitive Saturation (SILCS) algorithm was used to predict in silico candidate smal molecules that might be capable of binding at three diferent sites on HIF-2α that were identified by the algorithm based on the publicly available x- ray crystal structure of the HIF-2α:HIF-1β complex (PDB 4ZP4).

[0126] The top 100 candidate compounds at each of the three sites identified by SILCS (FIG.1A- C) were purchased commercialy (293 of 300 were available) and tested in HIF-1- and HIF-2- dependent cel-based reporter assays for their ability to inhibit HIF-1 and / or HIF-2 transcriptional activity. One compound that was predicted to bind at site 1 (compound refered to herein as “SS1.21”) and one compound that was predicted to bind at site 3 (compound refered to herein as 19 43011.601_P18269-03“SS3.2”) were identified as inhibitors of both HIF-1 and HIF-2. These compounds were found to inhibit the expression of endogenous HIF-1 and HIF-2 target genes (CA9 and EPO, respectively) in Hep3B human hepatocelular carcinoma cels with IC50 values of 0.3-1.6 micromolar (FIG.1E- FIG.1F).

[0127]

[0128] A secondary list of candidate compounds based on the structure of SS3.2 was produced, which were again tested in the same cel-based reporter assays. One compound (refered to herein as “3.2.16”) was found to inhibit the expression of endogenous HIF-1 and HIF-2 target genes in multiple cancer cel lines more potently than SS3.2. Other analogs of SS1.21, SS3.2, and 3.2.16 also were synthesized. One compound, referred to herein as “1.21.S9N” was found to have improved potency in vitro and greater bioavailability in vivo compared to SS1.21.

[0130] These chemical compounds (i.e., SS1.21, 1.21.S9N, SS3.2, and 3.2.16) were found to inhibit HIF target gene expression in more than 40 human and mouse cancer cel lines with IC50 values as low as 60 nM (FIG.3P). These compounds also safely inhibited the growth of human and mouse cancers in vivo. Further, in seven different tumor models, administration of 1.21.S9N or 3.2.16 in combination with immunotherapy (e.g., anti-CTLA4 or anti-PD1 antibody) led to complete tumor eradication in 24 out of 45 mice (53%) compared to 0 out of 45 vehicle- or isotype control-treated mice and 1 out of 30 mice (3.3%) treated with immunotherapy alone (Table 1). Thus, treatment with 1.21.S9N or 3.2.16 improved the rate of complete response to immunotherapy by more than 20-fold. Mice with tumor eradication were re-injected with the same 20 43011.601_P18269-03tumor cels and no tumors developed, whereas tumors formed rapidly in naive mice. In addition, administration of SS1.21 or 3.2.16 by daily intraperitoneal or single intravitreal injection, respectively, was shown to be efective at inhibiting laser-induced choroidal neovascularization, which is a mouse model of the neovascular (“wet”) type of age-related macular degeneration.

[0131] More particularly, in some embodiments, the presently disclosed subject mater provides a compound of formula (I): ;

[0134] is a double bond that can be present or absent;

[0135] L1 is a linker selected from –(CH2)t-, -O-CH2-CH2-, and -CH2-CH2-O-; wherein t is an integer selected from 1, 2, and 3;

[0136] A1 and A2 are each independently -CH- or N;

[0137] B1 and B2 can be present or absent and when present are each independently selected from -CR1R2- and -C(C=O)-, wherein R1 and R2 are each independently H or C1-C4 alkyl;

[0138] X1 and X2 are each independently selected from S, O, and SR3, wherein R3 is C1-C4 alkyl;

[0139] Y1 and Y2 are each independently selected from -CH2-, -NR4-, and -O-, wherein R4 is C1- C4 alkyl, or Y1 is -N- when X1 is -SR3 and / or Y2 is -N- when X2 is -SR3.

[0140] Z1 and Z2 are each independently selected from C1-C4 alkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl; and

[0141] stereoisomers and pharmaceuticaly acceptable salts thereof.

[0142] In certain embodiments, L1 is -(CH2)3- and the compound of formula (I) is a compound of formula (Ia): .(Ia) is selected from:21 43011.601_P18269-03i). endently S or O and the compound of formula (Ia-i) and compound of formula (Ia-i) are: of formula (Ia-a) and compound offormula (Ia-i) are:independently selected from substituted or unsubstituted C1-C8 straight chain or branched alkyl, phenyl, pyrimidinyl, imidazoyl, pyrazinyl, and pyridazinyl.

[0154] In certain embodiments, Z1 and Z2 are each independently selected from: 22 43011.601_P18269-03;

[0157] n is an integer selected from 0, 1, 2, 3, 4, and 5;

[0158] m is an integer selected from 0, 1, 2, 3, and 4;

[0159] p is an integer selected from 0, 1, 2 and 3; and

[0160] q is an integer selected from 0, 1, and 2; and

[0161] each R5 is independently selected from halogen, C1-C4 alkyl, hydroxyl, alkoxyl, cyano, - CF3, -NO2, -C(=O)H, -SR6, -SO2R7, -C(=O)-R8, and -C(=O)-O-R9, wherein R6, R7, R8, and R9 are independently C1-C4 alkyl.

[0162] In particular embodiments, the compound of formula (I) is selected from a compound listed in Table 10. 23 43011.601_P18269-03)I( alu mroFfo sdnuo e p r m ut o c C ur e t vi S t l at a n ci es m e e rp h e C R .01 elba T 24 43011.601_P18269-03)I( alu mroFf o sdnuo e p r m ut o c C ur e t vi S t l at a n ci es m e e rp h e C R .01 elba T 25 43011.601_P18269-03)I( alu mroFf o sdnuo e p r m ut o c C ur e t vi S t l at a n ci es m e e rp h e C R .01 elba T 26 43011.601_P18269-03)I(al S S S u mroFf o sdnuo e p r m ut o c C ur e t vi S t l at a n ci es m e e rp h e C R .01 elba T 27 43011.601_P18269-03)I( alu mroFf o sdnuo e p r m ut o c C ur e t vi S t l at a n ci es m e e rp h e C R .01 elba T 28 43011.601_P18269-03)I( alu mroFf o sdnuo e p r m ut o c C ur e t vi S t l at a n ci es m e e rp h e C R .01 elba T 29 43011.601_P18269-03)I( alu mroFf o sdnuo e p r m ut o c C ur e t vi S t l at a n ci es m e e rp h e C R .01 elba T 30 43011.601_P18269-03) I e I S r .1 .1 .1 ( c S S S S al S S S u mroFf o sdnuo e p r m ut o c C ur e t vi S t l at a n ci es m e e rp h e C R .01 elba T 31 43011.601_P18269-03)I( S S al S S S S S u mroFf o sdnuo e p r m ut o c C ur e t vi S t l at a n ci es m e e rp h e C R .01 elba T 32 43011.601_P18269-03)I(al S S S u mroFf o sdnuo e p r m ut o c C ur e t vi S t l at a n ci es m e e rp h e C R .01 elba T 33 43011.601_P18269-03)I( alu mroFf o sdnuo e p r m ut o c C ur e t vi S t l at a n ci es m e e rp h e C R .01 elba T 34 43011.601_P18269-03)I( alu mroFf o sdnuo e p r m ut o c C ur e t vi S t l at a n ci es m e e rp h e C R .01 elba T 35 43011.601_P18269-03)I( alumroFf o sdnuo e p r m ut o c C ur e t vi S t l at a n ci es m e e rp h e C R .01 elba T 36 43011.601_P18269-03)I( alu mroFf o sdnuo e p r m ut o c C ur e t vi S t l at a n ci es m e e rp h e C R .01 elba T 37 43011.601_P18269-03)I( alumroFf o sdnuo e p r m ut o c C ur e t vi S t l at a n ci es m e e rp h e C R .01 elba T 38 43011.601_P18269-03)I( alu mroFf o sdnuo e p r m ut o c C ur e t vi S t l at a n ci es m e e rp h e C R .01 elba T 39 43011.601_P18269-03)I( alu mroFf o sdnuo e p r m ut o c C ur e t vi S t l at a n ci es m e e rp h e C R .01 elba T 40 43011.601_P18269-03)I( alu mroFf o sdnuo e p r m ut o c C ur e t vi S t l at a n ci es m e e rp h e C R .01 elba T 41 43011.601_P18269-03)I( alu mroFf o sdnuo e p r m ut o c C ur e t vi S t l at a n ci es m e e rp h e C R .01 elba T 42 43011.601_P18269-03)I( alumroFf o sdnuo e p r m ut o c C ur e t vi S t l at a n ci es m e e rp h e C R .01 elba T 43 43011.601_P18269-03)I( alumroFf o sdnuo e p r m ut o c C ur e t vi S t l at a n ci es m e e rp h e C R .01 elba T 44 43011.601_P18269-03)I( alu mroFf o sdnuo e p r m ut o c C ur e t vi S t l at a n ci es m e e rp h e C R .01 elba T 45 43011.601_P18269-03)I( alu mroFf o sdnuo e p r m ut o c C ur e t vi S t l at a n ci es m e e rp h e C R .01 elba T 46 43011.601_P18269-03)I( alu mroFf o sdnuo e p r m ut o c C ur e t vi S t l at a n ci es m e e rp h e C R .01 elba T 47 43011.601_P18269-03)I( alu mroFf o sdnuo e p r m ut o c C ur e t vi S t l at a n ci es m e e rp h e C R .01 elba T 48 43011.601_P18269-03)I( alu mroFf o sdnuo e p r m ut o c C ur e t vi S t l at a n ci es m e e rp h e C R .01 elba T 49 43011.601_P18269-03)I( alu mroFf o sdnuo e p r m ut o c C ur e t vi S t l at a n ci es m e e rp h e C R .01 elba T 50 43011.601_P18269-03no 1 P is M 4.2 )I( alu mr mo rF of F o f o sd s n d u n o e u e p r o r m ut p u o c m tc C u o r u e t C r vi S e t l vi Stat a t l c a an i t ce is m nee e s mr h e ep r C p he e C R R .0 . 1 01el eb la b T a T 51 43011.601_P18269-03)I( alu mroFf o sdnuo e p r m u o tc C u e rt vi S ta l t a n c e i se m r e p h e C R .01 elba T 52 43011.601_P18269-03)I(a x l e H .4 H µ u mroFf o sdnuo e p r m u o tc C u e rt vi S ta l t a n c e i se m r e p h e C R .01 elba T 53 43011.601_P18269-03)I( alu mroFfo sdnuo e p r m u o tc C u e rt vi S ta l t a n c e i se m r e p h e C R .01 elba T 54 43011.601_P18269-03) e e I r C G p I ( x e n alu mroFf o sdnuo e p r m u o tc C u e rt vi S ta l t a n c e i se m r e p h e C R .01 elba T 55 43011.601_P18269-03o e is 0 n se 5 ) e C I r G p I ( x n al e u mroFf o sdnuo e p r m u o tc C u e rt vi S ta l t a n c e i se m r e p h e C R .01 elba T 56 43011.601_P18269-03o e is 0 n se 5 ) e C I r G p I ( x n al e u mroFf o sdnuo e p r m u o tc C u e rt vi S ta l t a n c e i se m r e p h e C R .01 elba T 57 43011.601_P18269-03)I( alu mroFf o sdnuo e p r m u o tc C u e rt vi S ta l t a n c e i se m r e p h e C R .01 elba T 58 43011.601_P18269-03)I( alu mroFf o sdnuo e p r m u o tc C u e rt vi S ta l t a n c e i se m r e p h e C R .01 elba T 59 43011.601_P18269-03es n s 5 e e )I r CI ( G px n al e u mroFf o sdnuo e p r m u o tc C u e rt vi S ta l t a n c e i se m r e p h e C R .01 elba T 60 43011.601_P18269-03es n s 5 e e )I r CI ( G px n al e u mroFf o sdnuo e p r m u o tc C u e rt vi S ta l t a n c e i se m r e p h e C R .01 elba T 61 43011.601_P18269-03e s n s 05 ) e e I r CI ( G p n a x l e u mroFf o sdnuo e p r m u o tc C u e rt vi S ta l t a n c e i se m r e p h e C R .01 elba T 62 43011.601_P18269-03en se 5 ) e I r C ( G p I x e n alu mroFf o sdnuo e p r m u o tc C u e rt vi S ta l t a n c e i se m r e p h e C R .01 elba T 63 43011.601_P18269-03e s n s 05 ) e e I r CI ( G p n a x l e u mroFf o sdnuo e p r m u o tc C u e rt vi S ta l t a n c e i se m r e p h e C R .01 elba T 64 43011.601_P18269-03n se ) e C I rp I ( G x e n alu mroFf o sdnuo e p r m u o tc C u e rt vi S ta l t a n c e i se m r e p h e C R .01 elba T 65 43011.601_P18269-03es n s 5 e e )I r CI ( G px n al e u mroFf o sdnuo e p r m u o tc C u e rt vi S ta l t a n c e i se m r e p h e C R .01 elba T 66 43011.601_P18269-03) e e I r C G p I ( x e n alu mroFf o sdnuo e p r m u o tc C u e rt vi S ta l t a n c e i se m r e p h e C R .01 elba T 67 43011.601_P18269-03e s n s 05 ) e e I r CI ( G p n a x l e u mroFf o sdnuo e p r m u o tc C u e rt vi S ta l t a n c e i se m r e p h e C R .01 elba T 68 43011.601_P18269-03es n s 5 e e )I r CI ( G px n al e u mroFf o sdnuo e p r m u o tc C u e rt vi S ta l t a n c e i se m r e p h e C R .01 elba T 69 43011.601_P18269-03n) e e C I r G p I ( x e n alu mroFf o sdnuo e p r m u o tc C u e rt vi S ta l t a n c e i se m r e p h e C R .01 elba T 70 43011.601_P18269-03)I( alu mroFf o sdnuo e p r m u o tc C u e rt vi S ta l t a n c e i se m r e p h e C R .01 elba T 71 43011.601_P18269-03e)I r I ( G px n al e u mroFf o sdnuo e p r m u o tc C u e rt vi S ta l t a n c e i se m r e p h e C R .01 elba T 72 43011.601_P18269-03)I(n a x l e u mroFf o sdnuo e p r m u o tc C u e rt vi S ta l t a n c e i se m r e p h e C R .01 elba T 73 43011.601_P18269-03)I( G px e n alu mroFf o sdnuo e p r m u o tc C u e rt vi S ta l t a n c e i se m r e p h e C R .01 elba T 74 43011.601_P18269-03) eI r CI ( G p n a x l e u mroFf o sdnuo e p r m u o tc C u e rt vi S ta l t a n c e i se m r e p h e C R .01 elba T 75 43011.601_P18269-03)I p I ( G x e n alu mroFf o sdnuo e p r m u o tc C u e rt vi S ta l t a n c e i se m r e p h e C R .01 elba T 76 43011.601_P18269-03n) e e C I r G p I ( x e n alu mroFf o sdnuo e p r m u o tc C u e rt vi S ta l t a n c e i se m r e p h e C R .01 elba T 77 43011.601_P18269-03

[0163] In more particular embodiments, the compound of formula (I) is selected from: ; provides a compound offormula (I): ;

[0170] L2 is a linker which can be present or absent and when present is selected from: ;an integer selected from 0, 1, 2, and 3, each X3 can be present or absent and when present is -O-, and R13 is selected from H, C1-C4 alkyl, and -NR14R15, wherein R14 and R15 are each H or C1-C4 alkyl;

[0173] R10 is selected from a substituted or unsubstituted cycloalkyl or heterocycloalkyl, straight chain or branched C1-C4 alkyl, C1-C4 alkoxyl, -C(=O)-O-R16, and -CH2-C(=O)-O-R17, wherein R16 and R17 are each independently C1-C4 alkyl;

[0174] R11 is selected from H, straight chain or branched C1-C4 alkyl, -NR18R19, wherein R18 and R19 are each H or C1-C4 alkyl;

[0175] R12 is selected from H and halogen; or

[0176] R10 and R12 together form a 5- to 6- membered cycloalkyl or heterocycloalkyl ring;

[0177] Z3 is selected from C1-C4 alkyl, aryl, and heteroaryl, each of which can be substituted or unsubstituted; and

[0178] stereoisomers and pharmaceuticaly acceptable salts thereo. 78 43011.601_P18269-03

[0179] In certain embodiments, ring C is selected from: . formula (I) is selected from:or unsubstituted cycloalkyl or heterocycloalkyl selected from: ;

[0187] R20 is selected from H, C1-C4 alkyl, hydroxyl, and -O-C(=O)-R24, wherein R24 is C1-C4 alkyl;

[0188] R21 is H or C1-C4 alkyl;

[0189] R22 is selected from H, C1-C4 alkyl, and phenyl; or

[0190] R20 and R21 together form a 1,3-dioxolane ring structure.

[0191] In certain embodiments, Z3 is selected from: 79 43011.601_P18269-03;

[0194] n3 is an integer selected from 0, 1, 2, 3, 4, and 5;

[0195] m1 is an integer selected from 0, 1, 2, 3, and 4;

[0196] p1 is an integer selected from 0, 1, 2 and 3; and

[0197] q1 is an integer selected from 0, 1, and 2; and

[0198] each R25 is independently selected from halogen, C1-C4 alkyl, hydroxyl, alkoxyl, cyano, - CF3, -NO2, -C(=O)H, -SR26, -SO2R27, -C(=O)-R28, and -C(=O)-O-R29, wherein R26, R27, R28, and R29 are independently C1-C4 alkyl.

[0199] In particular embodiments, the compound of formula (I) is selected from a compound listed in Table 11. 80 43011.601_P18269-035 U 1 a 7 S M - 1 . 5 a 5 9 M 3 : M µ U 1 1 . S M - 9 M 4 : M µ )II(al - 13 - 0 u H H H 3 m Y D I Y 63 4 Y 63 8roF e 2f C e . 2. o L r DI 3 3 s I d S S c S n S S S S n nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 81 43011.601_P18269-0359 4 M W 3 2 ) 3 II( al - 3 - 7 u H H 46 H 4 m Y D I Y 3 2 Y 63 6roF e 2. 2f C e D 3 r .3 r o L s I rc I S e e d S S S n S d - S S d -nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 82 43011.601_P18269-0325 7 M W 3 6 ) 3 II( al - 7 - 8 u H H 46 H 4 m Y D I Y 3 8 Y 63 0roF e 2. 2f C e D 3 r .3 r o L s I rc I S e e d S S S n S d - S S d -nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 83 43011.601_P18269-0376 2 M W 3 5 ) 3 II( al - 8 - 8 u H H 46 H 4 m Y D I Y 3 2 Y 63 4roF e 2. 2f C e D 3 r .3 r o L s I rc I S e e d S S S n S d - S S d -nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 84 43011.601_P18269-035 a 5 U 1 1 . S M - 9 M 0 : M µ)II( al - 8 - 0u H H 4 H 5 Y D 6 6 m I Y 3 6 Y 3 0roFf C ee 2. 2. o L s I r DI 3 re 3 d S S c S n S S d - S S t n nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 85 43011.601_P18269-0362 6 M W 3 0 ) 4 II( al - 0 - 9 u H H 56 H 5 m Y D I Y 3 2 Y 63 0roF e 2. 2f C e D 3 r .3 r o L s I rc I S e e d S S S n S d - S S d -nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 86 43011.601_P18269-03M 1 5 ) W 4 3 II( al - 9 - 9 u H D H 56 H 56 m Y I Y 3 4 Y 3 6roF e 2f C e . 2. r D 3 r 3 r o L s I c I S e S e d S S S n S d - S d -nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 87 43011.601_P18269-035 a 4 U 1 1 . S M - 9 M 2 : M µ) 3 3 II( al - 9 u H 5 D H Y 6 m I Y 3 8 roFf C e o L e 2. 2. r DI 3 re 3 7 s I d S S c S n S S d - S S 6. nuo O p moc evita N t 25 ne e . s r 2: e u N P r t N gp c e ur o L R t S . l N1 a 1 ci el m b e a h O T C H 88 43011.601_P18269-035 a 4 U 1 1 . S M - 9 M 3 : M µ)II( al - 94 - 8u H H H 4 Y D I 6 6 m Y 3 0 Y 3 8roFf C ee 2. 2 n . - o L s I rc DI 3S 7 3 7 r d S S S n S 6. S S 6. e d nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 89 43011.601_P18269-0321 2 ) M W 3 13 II( al - 0 - 0 u H H 56 H 56 m Y D I Y 3 4 Y 3 6roFf C ee 2. 2. r D 3 - r 3 - o L s I c I S 7 7 r d S S S n S 6. e d S S 6. e d nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 90 43011.601_P18269-0392 9 ) M W 3 23 II( al - 0 - 1 u H H 56 H 56 m Y D I Y 3 8 Y 3 0roFf C ee 2. 2. r D 3 - r 3 - o L s I c I S 7 7 r d S S S n S 6. e d S S 6. e d nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 91 43011.601_P18269-0337 0 ) M W 3 33 II( al - 1 - 1 u H H 56 H 56 m Y D I Y 3 2 Y 3 4roFf C ee 2. 2. r D 3 - r 3 - o L s I c I S 7 7 r d S S S n S 6. e d S S 6. e d nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 92 43011.601_P18269-03M 2 2 ) W 3 3 II( al - 15 - 1u H H H 5 Y D 6 6 m I Y 3 6 Y 3 8roFf C ee 2. 2. o L s I rc DI 3 - S 7 r 3 - S 7 r d S S S n S 6. e d S 6. e d nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 93 43011.601_P18269-03M 2 6 ) W 3 3 II( al - 25 - 2u H H H 5 Y D 6 6 m I Y 3 0 Y 3 4roFf C ee 2. 2. o L s I rc DI 3 - S 7 r 3 - S 7 r d S S S n S 6. e d S 6. e d nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 94 43011.601_P18269-03M 2 1 ) W 3 3 II( al - 25 - 2u H H H 5 Y D 6 6 m I Y 3 6 Y 3 8roFf C ee 2. 2. o L s I rc DI 3 - S 7 r 3 - S 7 r d S S S n S 6. e d S 6. e d nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 95 43011.601_P18269-0380 8 ) M W 3 03 II( al - 3 - 3 u H H 56 H 56 m Y D I Y 3 0 Y 3 2roFf C ee 2. 2. r D 3 - r 3 - o L s I c I S 7 7 r d S S S n S 6. e d S S 6. e d nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 96 43011.601_P18269-0393 9 ) M W 3 33 II( al - 3 - 3 u H H 56 H 56 m Y D I Y 3 6 Y 3 8roFf C ee 2. 2. r D 3 - r 3 - o L s I c I S 7 7 r d S S S n S 6. e d S S 6. e d nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 97 43011.601_P18269-035 U 1 a 1 S M - 1 . 9 M 0 : M µ) M W 3 3 II( al - 4 - 0 u H D H 56 H 66 m Y I Y 3 0 Y 3 0roFf C ee 2. 2 - . o L s I r DI 3 7 r 3 6 d S S c S n S S 6. e d S S 1. nuop moc evitatne e s r O e u r t p cu N e r R t N S . l 1 a N1 ci el m b e a h O T C H 98 43011.601_P18269-035 a 2 U 1 1 . S M - 9 M 2 : M µ)II( al - 35 - 8u H H H 5 Y D I 6 6 m Y 3 4 Y 3 8roFf C ee 2. 2 n . - o L s I rc DI 3S 6 3 6 r d S S S n S 1. S S 1. e d nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 99 43011.601_P18269-03)II( alu mroFf C. . . o L er DI 3 -6 r 3 - r 3 - rs I c S S 6 S 6 d S S S n S 1. e d S 1. e d S 1. e dnuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 100 43011.601_P18269-035 a 5 U 1 1 . S M - 9 M 1 : M µ )II( al - 16 - 2u H H H 6 Y D I 6 6 m Y 3 2 Y 3 6roFf C ee 2. c 2 a . o L s I rc DI 3S 6 3 -6 r d S S S n S 1. S S 1. e d nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 101 43011.601_P18269-0397 9 ) M W 2 03 II( al - 5 - 6 u H H 66 H 66 m Y D I Y 3 8 Y 3 0roFf C ee 2. 2. r D 3 - r 3 - o L s I c I S 6 6 r d S S S n S 1. e d S S 1. e d nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 102 43011.601_P18269-0348 4 ) M W 2 82 II( al - 6 - 6 u H H 66 H 66 m Y D I Y 3 2 Y 3 4roFf C ee 2. 2. r D 3 - r 3 - o L s I c I S 6 6 r d S S S n S 1. e d S S 1. e d nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 103 43011.601_P18269-0348 5 ) M W 2 23 II( al - 6 - 7 u H H 66 H 66 m Y D I Y 3 6 Y 3 8roFf C ee 2. 2. r D 3 - r 3 - o L s I c I S 6 6 r d S S S n S 1. e d S S 1. e d nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 104 43011.601_P18269-0348 5 ) M W 2 23 II( al - 8 - 9 u H H 66 H 66 m Y D I Y 3 8 Y 3 0roFf C ee 2. 2. r D 3 - r 3 - o L s I c I S 6 6 r d S S S n S 1. e d S S 1. e d nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 105 43011.601_P18269-0352 7 ) M W 3 33 II( al - 9 - 1 u H H 66 H 76 m Y D I Y 3 2 Y 3 4roFf C ee 2. 2. r D 3 - r 3 - o L s I c I S 6 6 r d S S S n S 1. e d S S 1. e d nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 106 43011.601_P18269-0373 0 ) M W 3 23 II( al - 1 - 1 u H H 76 H 76 m Y D I Y 3 6 Y 3 8roFf C ee 2. 2. r D 3 - r 3 - o L s I c I S 6 6 r d S S S n S 1. e d S S 1. e d nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 107 43011.601_P18269-0373 1 ) M W 3 53 II( al - 2 - 4 u H H 76 H 76 m Y D I Y 3 2 Y 3 6roFf C ee 2. 2. r D 3 - r 3 - o L s I c I S 6 6 r d S S S n S 1. e d S S 1. e d nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 108 43011.601_P18269-0392 4 ) M W 3 82 II( al - 4 - 5 u H H 76 H 76 m Y D I Y 3 8 Y 3 0roFf C ee 2. 2. r D 3 - r 3 - o L s I c I S 6 6 r d S S S n S 1. e d S S 1. e d nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 109 43011.601_P18269-0335 7 ) M W 2 62 II( al - 5 - 5 u H H 76 H 76 m Y D I Y 3 2 Y 3 4roFf C ee 2. 2. r D 3 - r 3 - o L s I c I S 6 6 r d S S S n S 1. e d S S 1. e d nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 110 43011.601_P18269-0318 7 ) M W 2 92 II( al - 5 - 6 u H H 76 H 76 m Y D I Y 3 6 Y 3 0roFf C ee 2. 2. r D 3 - r 3 - o L s I c I S 6 6 r d S S S n S 1. e d S S 1. e d nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 111 43011.601_P18269-0338 1 ) M W 2 03 II( al - 6 - 8 u H H 76 H 76 m Y D I Y 3 2 Y 3 4roFf C ee 2. 2. r D 3 - r 3 - o L s I c I S 6 6 r d S S S n S 1. e d S S 1. e d nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 112 43011.601_P18269-0310 1 ) M W 3 03 II( al - 8 - 8 u H H 76 H 76 m Y D I Y 3 6 Y 3 8roFf C ee 2. 2. r D 3 - r 3 - o L s I c I S 6 6 r d S S S n S 1. e d S S 1. e d nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 113 43011.601_P18269-0381 8 ) M W 3 13 II( al - 0 - 0 u H H 86 H 86 m Y D I Y 3 2 Y 3 4roFf C ee 2. 2. r D 3 - r 3 - o L s I c I S 6 6 r d S S S n S 1. e d S S 1. e d nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 114 43011.601_P18269-0381 2 ) M W 3 63 II( al - 0 - 1 u H H 86 H 86 m Y D I Y 3 6 Y 3 4roFf C ee 2. 2. r D 3 - r 3 - o L s I c I S 6 6 r d S S S n S 1. e d S S 1. e d nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 115 43011.601_P18269-0326 7 ) M W 3 92 II( al - 1 - 5 u H H 86 H 96 m Y D I Y 3 6 Y 3 4roFf C ee 2. 2. r D 3 - r 3 - o L s I c I S 6 6 r d S S S n S 1. e d S S 1. e d nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 116 43011.601_P18269-0352 5 ) M W 3 82 II( al - 5 - 7 u H H 96 H 96 m Y D I Y 3 8 Y 3 4roFf C ee 2. 2. r D 3 - r 3 - o L s I c I S 6 6 r d S S S n S 1. e d S S 1. e d nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 117 43011.601_P18269-0332 7 ) M W 3 23 II( al - 7 - 8 u H H 96 H 96 m Y D I Y 3 8 Y 3 0roFf C ee 2. 2. r D 3 - r 3 - o L s I c I S 6 6 r d S S S n S 1. e d S S 1. e d nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 118 43011.601_P18269-0373 5 ) M W 3 92 II( al - 8 - 8 u H H 96 H 96 m Y D I Y 3 6 Y 3 8roFf C ee 2. 2. r D 3 - r 3 - o L s I c I S 6 6 r d S S S n S 1. e d S S 1. e d nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 119 43011.601_P18269-0379 5 ) M W 2 92 II( al - 9 - 9 u H H 96 H 96 m Y D I Y 3 0 Y 3 2roFf C ee 2. 2. r D 3 - r 3 - o L s I c I S 6 6 r d S S S n S 1. e d S S 1. e d nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 120 43011.601_P18269-0311 8 ) M W 3 92 II( al - 9 - 9 u H H 96 H 96 m Y D I Y 3 4 Y 3 8roFf C ee 2. 2. r D 3 - r 3 - o L s I c I S 6 6 r d S S S n S 1. e d S S 1. e d nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 121 43011.601_P18269-0390 5 ) M W 3 92 II( a - 0 - 1 )l 0 0 Ru H H H ( Y D I Y 73 2 Y 7 -m 3 8roF e 2 2f C e D .3 - .3 - o L s I r I 6 r 6 r d S S c S n S S 1. e d S S 1. e d nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 122 43011.601_P18269-0359 4 ) M W 2 33 II( al - 1 ) - 2u H H 0 S( H 0 Y D I Y 73 - 8 Y 7m 3 6roF e 2 2f C e D .3 - .3 - o L s I r I 6 r 6 r d S S c S n S S 1. e d S S 1. e d nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 123 43011.601_P18269-0303 7 ) M W 2 43 II( al - 2 - 3 u H H 07 H 07 m Y D I Y 3 8 Y 3 8roFf C ee 2. 2. r D 3 - r 3 - o L s I c I S 6 6 r d S S S n S 1. e d S S 1. e d nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 124 43011.601_P18269-03M 2 0 ) W 3 3 II( al - 40 - 6u H H H 0 Y D 7 7 m I Y 3 0 Y 3 8roFf C ee 2. 2. o L s I rc DI 3 - S 6 r 3 - S 6 r d S S S n S 1. e d S 1. e d nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 125 43011.601_P18269-0351 6 ) M W 3 82 II( al - 7 - 7 u H H 07 H 07 m Y D I Y 3 0 Y 3 2roFf C ee 2. 2. r D 3 - r 3 - o L s I c I S 6 6 r d S S S n S 1. e d S S 1. e d nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 126 43011.601_P18269-03M 8 9 ) W 2 2 II( al - 70 - 7u H H H 0 Y D 7 7 m I Y 3 4 Y 3 6roFf C ee 2. 2. o L s I rc DI 3 - S 6 r 3 - S 6 r d S S S n S 1. e d S 1. e d nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 127 43011.601_P18269-0358 3 ) M W 2 14 II( al - 7 - 8 u H H 07 H 07 m Y D I Y 3 8 Y 3 4roFf C ee 2. 2. r D 3 - r 3 - o L s I c I S 6 6 r d S S S n S 1. e d S S 1. e d nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 128 43011.601_P18269-0396 7 ) M W 3 23 II( al - 9 - 9 u H H 07 H 07 m Y D I Y 3 4 Y 3 8roFf C ee 2. 2. r D 3 - r 3 - o L s I c I S 6 6 r d S S S n S 1. e d S S 1. e d nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 129 43011.601_P18269-03)II( alu mroFfo LI rc I S 6 re S 6 r S 6 rsd S S S n S 1. d S 1. e d S 1. e dnuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 130 43011.601_P18269-0392 5 ) M W 3 23 II( al - 41 6 1 - 1u H H H 1 Y D I 7 7 m Y 3 Y 3roFf C ee 2. 2. r D 3 - r 3 - o L s I c I S 6 6 r d S S S n S 1. e d S S 1. e d nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 131 43011.601_P18269-03L a y I oiti c y u a b 5 s ih ta % L s a n i n M μ 81 go 6 l P .3 21 ) M W 3 II( al - 81u H H 1 Y D I 7 m Y 3 roFf C ee 2 D .3 - o L r I 6 r s I d S S c S n S S 1. e d nuop moc evitatne e s r e u r t p c e ur R t S . l 1 a 1 ci el m b e a h T C 132 43011.601_P18269-03

[0200] In more particular embodiments, the compound of formula (I) is selected from: .comprising a compound of formula (I) or formula (I) and an pharmaceuticaly acceptable carier. In particular embodiments, formulation comprises a liposomal composition, niosomes, nanoemulsions, nanosuspensions, miceles, including nanomiceles, hydrogels, polymeric nanoparticles, including PLGA nanoparticles and other biocompatible polymeric nanoparticles, solid lipid nanoparticles, nanostructured lipid cariers, nanocrystals, dendrimers, cubosomes, olaminosomes, bilosome, extracelular vesicles (exosomes), and smart nano-micro platforms.

[0204] In certain embodiments, the formulation comprises a liposomal composition. Representative liposomal compositions are disclosed in U.S. provisional patent application no. 63 / 644,289, for LIPOSOMAL NANOPARTICLES CONTAINING HIF INHIBITOR 32-134D FOR TREATMENT OF OCULAR NEOVASCULARIZATION, to Semenza et al., filed May 8, 2024, atorney docket no.: JHU-42735.101 (C18152_P18152-01), which is incorporated herein by reference in its entirety.

[0205] As used herein, liposomal nanoparticles are concentric bilayer vesicles mainly composed of phosphatidylcholine or other phospholipid with or without cholesterol. Liposomes can be characterized into four categories based on size and number of bilayers: smal unilamelar vesicles (SUV), large unilamelar vesicles (LUV), multilamelar vesicle (MLV), and multivesicular vesicles (MVV). In certain embodiments, the presently claimed LNPs are smal unilamelar vesicles (SUV).

[0206] Liposomes can be prepared from natural lipids or synthetic lipids. Natural lipids, including natural phospholipids, can be obtained from various sources, such as soya bean and egg yolk. Natural phospholipids suitable for use with the presently disclosed subject mater include, but are 133 43011.601_P18269-03not limited to, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), and phosphatidic acid (PA).

[0207] Synthetic lipids suitable for use with the presently disclosed subject mater include, but are not limited to: palmitic acid-based synthetic phospholipids including, but not limited to, 1,2- dipalmitoyl-sn-glycero-3-phosphorylethanolamine (DPPE), 1,2-dipalmitoyl-sn-glycero-3- phosphatidic acid sodium salt (DPPA), 1,2-dipalmitoyl-sn-glycero-3-phosphorylglycerol sodium salt (DPPG), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC); stearic acid-based synthetic phospholipids including, but not limited to, 1,2-distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE), 1,2-distearoyl-sn-glycero-3-phosphatidic acid Na salt (DSPA), 1,2-distearoyl-sn-glycero- 3-phosphorylglycerol sodium salt (DSPG), 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC); and other synthetic phospholipids including, but not limited to, 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP), L-a-phosphatidylcholine (HSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and 1,2-dimyristoyl-sn-glycero-3- phosphocholine (DMPC).

[0208] In certain embodiments, the LNPs comprise a lipid selected from egg-yolk phosphatidylcholine (ePC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2- Dioleoyl-sn-glycero-3-phosphocholine (DOPC). In particular embodiments, the lipid comprises ePC.

[0209] In some embodiments, the LNPs can include a steroid, such as cholesterol or DC- cholesterol. Such steroids can be used to improve the liposome rigidity, stability, fluidity, permeability, membrane strength, elasticity and stifness, transition temperature (Tm), drug retention, phospholipid packing, and the like. A steroid, such as cholesterol is typicaly present in a ratio less than about 30%, including 30, 25, 20, 25, 10, 5, 4, 3, 2, 1, and 0.5% of the total lipids. In certain embodiments, the presently disclosed LNPs further comprise cholesterol.

[0210] In some embodiments, the LNPs further comprise a pegylated lipid. Representative pegylated lipids include, but are not limited to, PEG350 PE, PEG550 PE, PEG750 PE, PEG1000 PE, PEG2000 PE, PEG 3000 PE, and PEG5000 PE, among others. In certain embodiments, the pegylated lipid comprises PEG2000 PE. In more certain embodiments, the pegylated lipid is selected from 14:0 PEG2000 PE, 16:0 PEG2000 PE, 18:0 PEG2000 PE, and 18:1 PEG2000 PE. 134 43011.601_P18269-03

[0211] In particular embodiments, the pegylated lipid comprises 1,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000 (18:0 PEG2000 PE) or a pharmaceuticaly acceptable salt thereof, having the folowing chemical structure: . is1:3:0.03; 1:3:0.05; 1:3:0.1; 1:3:0.15; 1:3:0.2; 1:3:0.3; 1:3:0.5; 1:3:0.6; 1.5:3:0.05; 2.0:3:0.05; 2:6:0.10; 3.0:3:0.05; 3:9:0.15; and 4:12:0.20. In particular embodiments, the lipid:HIF inhibitor:PEG is 1:3:0.2.

[0214] In certain embodiments, the presently disclosed compounds of formula (I) and formula (I) can be formulated as liposomes, niosomes, nanoemulsions, nanosuspensions, miceles, including nanomiceles, hydrogels, polymeric nanoparticles, including PLGA nanoparticles and other biocompatible polymeric nanoparticles, solid lipid nanoparticles, nanostructured lipid cariers, nanocrystals, dendrimers, cubosomes, olaminosomes, bilosome, extracelular vesicles (exosomes), and smart nano-micro platforms.

[0215] In some embodiments, the formulation comprises a compound of formula (I) or formula (I) incorporated a microparticle or microsphere comprising one or more biodegradable polymers including, but not limited to, a poly(lactic-co-glycolic)acid (PLGA), a poly(ε-caprolactone) (PCL), a poly(lactic acid) (PLA), a poly(glycolic acid) (PGA), a polyester, a poly(orthoester), a poly(phosphazine), a poly(phosphate ester), a poly(ε-caprolactone-co-ethyl ethylene phosphate) (PCLEEP), a polyvinyl alcohol (PVA), a poly(β-amino ester), a poly(acrylic acid) (PAA), a poly- 3-hydroxybutyrate (P3HB), a poly(hydroxybutyrate-co-hydroxyvalerate), a polyethylene glycol (PEG), and combinations thereof.

[0216] In some embodiments, the compound of formula (I) or formula (I) are formulated with a hydrogel. Suitable hydrogels for ocular drug delivery are reviewed in Lynch et al., Hydrogel Biomaterials for Application in Ocular Drug Delivery, Front Bioeng Biotechnol.2020; 8: 228.

[0217] In some embodiments, the hydrogel is an in situ geling system. In some embodiments, the hydrogel is a stimuli-responsive hydrogel, including temperature-sensitive hydrogel systems, 135 43011.601_P18269-03pH-sensitive hydrogel systems, such as those with carbopol (polyacrylic acid (PAA), polyethylene glycol, and natural biopolymers, and ion-sensitive hydrogel systems, e.g., an alginate hydrogel, ultrasound-responsive hydrogel systems. In some embodiments, the temperature- sensitive hydrogel system can comprise a PLGA–PEG–PLGA (PPP) triblock copolymer.

[0218] In other embodiments, iontophoresis can be used to enhance the penetration of an ocular active ingredient through the various tissue layers found in the eye.

[0219] In some embodiments, the hydrogel comprises a natural biopolymer including, but not limited to, chitosan, hyaluronic acid, gelatin, alginate, methylcelulose, and colagen.

[0220] The nanoparticles and microparticles of the presently disclosed subject mater can have a size ranges from about 1 nm to about 1000 nm for nanoparticles and from about 1 μm to about 1000 μm for the microparticles.

[0221] In some embodiments, nanoparticle can have a size ranging from about range of 1 to 1000 nm in size, including about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 600, 700, 800, 900, and 1000 nm, in size. In some embodiments, the microparticle or microsphere can have a size ranging from about range of 1 to 1000 μm in size, including about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 600, 700, 800, 900, and 1000 μm, in size.

[0222] METHODS OF TREATMENT

[0223] In other embodiments, the presently disclosed subject mater provides a method for treating a disease, disorder, or condition associated with one or more hypoxia inducible factors (HIFs) in a subject in need of treatment thereof, the method comprising administering to the subject a therapeuticaly efective amount of a compound of formula (I) or formula (I) or a formulation thereof.

[0224] In certain embodiments, the disease, disorder, or condition is a cancer. In particular embodiments, the cancer is selected from breast cancer, colorectal cancer, lung cancer, melanoma, pancreatic cancer, prostate cancer, and head and neck squamous cel carcinoma (HNSCC). In certain embodiments, administration of the compound of formula (I) or formula (I) inhibits or 136 43011.601_P18269-03blocks growth and / or vascularization of a tumor associated with breast cancer, colorectal cancer, lung cancer, melanoma, pancreatic cancer, or prostate cancer.

[0225] Other cancers potentialy treatable by the compounds formula (I) and formula (I) and formulations thereof include, but are not limited to, acute lymphoblastic leukemia (ALL), acute myeloid leukemia, adrenocortical carcinoma, adult acute, AIDS-related cancers, AIDS-related lymphoma, anal cancer, appendix cancer, arcoma, astrocytoma (childhood cerebelar or cerebral), barcinoid tumor, basal-cel carcinoma, bile duct cancer, bladder cancer bone tumor, brain cancer, brain tumor, brainstem glioma, breast cancer, bronchial adenomas / carcinoids, Burkit’s lymphoma, carcinoma of unknown primary, cerebelar astrocytoma, cerebral astrocytoma / malignant glioma, cervical cancer, childhood, childhood acute, childhood cerebral astrocytoma, childhood visual pathway and hypothalamic, chondrosarcoma, chronic, chronic lymphocytic, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloid leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous, cutaneous t-cel, cutaneous t-cel lymphoma, desmoplastic smal round cel tumor, endometrial, endometrial cancer, ependymoma, esophageal cancer, Ewing family of tumors, Ewing’s sarcoma, extracranial germ cel tumor, extragonadal germ cel tumor, extrahepatic bile duct cancer, extrahepatic cancer, galbladder cancer, gastric (stomach) cancer, gastric carcinoid, gastrointestinal, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (gist), germ cel tumor: extracranial, extragonadal, or ovarian, gestational, gestational trophoblastic tumor, glioma, glioma of the brain stem, hairy cel leukemia, hepatocelular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma, intraocular melanoma, islet cel carcinoma (endocrine pancreas), Kaposi sarcoma, kidney cancer (renal cel cancer), laryngeal cancer, leukaemias, lip and oral cavity cancer, liposarcoma, liver cancer (primary), lung cancer, lymphomas, macroglobulinemia, malignant fibrous histiocytoma of bone / osteosarcoma, meduloblastoma, melanoma, Merkel cel cancer, mesothelioma, metastatic, mouth cancer, multiple (cancer of the bone-marow), multiple endocrine neoplasia syndrome, multiple myeloma / plasma cel neoplasm, mycosis fungoides, myelodysplastic syndromes, myelodysplastic / myeloproliferative diseases, myelogenous leukemia, myeloid leukemia, myeloma, myeloproliferative disorders, myxoma, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma neuroblastoma, non-Hodgkin lymphoma, non-smal cel, non-smal cel lung cancer, oligodendroglioma, oral cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma, ovarian cancer, ovarian 137 43011.601_P18269-03epithelial cancer (surface epithelial-stromal tumor), ovarian germ cel tumor, ovarian low malignant potential tumor, pancreatic cancer, pancreatic cancer, islet cel, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary adenoma, plasma cel neoplasia / multiple myeloma, pleuropulmonary blastoma, primary central nervous system, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cel carcinoma, renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sezary syndrome, skin cancer (melanoma), skin cancer (non-melanoma), skin carcinoma, smal cel, smal cel lung cancer, smal intestine cancer, soft tissue, soft tissue sarcoma, squamous cel carcinoma, squamous neck cancer with occult primary, stomach cancer, supratentorial primitive neuroectodermal tumors, t-cel lymphoma, testicular cancer, throat cancer, thymoma, thymoma and thymic carcinoma, thyroid cancer, transitional cel cancer, transitional cel cancer of the renal pelvis and ureter, trophoblastic tumor, unknown primary site, ureter and renal pelvis, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma, vulvar cancer, Waldenstrom, Waldenstrom macroglobulinemia, and Wilms tumor (kidney cancer).

[0226] In other embodiments, the method further comprises administering the compound of formula (I) or formula (I) in combination with an immunotherapy. In certain embodiments, the immunotherapy comprises immune checkpoint blockade (ICB) immunotherapy. In particular embodiments, the ICB immunotherapy includes anti-CTLA-4, anti-PD-1, or anti-PD-L1 immunotherapy.

[0227] In particular embodiments, administering the compound of formula (I) or formula (I) in combination with the immunotherapy alters an immune cel microenvironment of a tumor associated with acute lymphoblastic leukemia (ALL), acute myeloid leukemia, adrenocortical carcinoma, adult acute, AIDS-related cancers, AIDS-related lymphoma, anal cancer, appendix cancer, arcoma, astrocytoma (childhood cerebelar or cerebral), barcinoid tumor, basal-cel carcinoma, bile duct cancer, bladder cancer bone tumor, brain cancer, brain tumor, brainstem glioma, breast cancer, bronchial adenomas / carcinoids, Burkit’s lymphoma, carcinoma of unknown primary, cerebelar astrocytoma, cerebral astrocytoma / malignant glioma, cervical cancer, childhood, childhood acute, childhood cerebral astrocytoma, childhood visual pathway and hypothalamic, chondrosarcoma, chronic, chronic lymphocytic, chronic lymphocytic leukemia, 138 43011.601_P18269-03chronic myelogenous leukemia, chronic myeloid leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous, cutaneous t-cel, cutaneous t-cel lymphoma, desmoplastic smal round cel tumor, endometrial, endometrial cancer, ependymoma, esophageal cancer, Ewing family of tumors, Ewing’s sarcoma, extracranial germ cel tumor, extragonadal germ cel tumor, extrahepatic bile duct cancer, extrahepatic cancer, galbladder cancer, gastric (stomach) cancer, gastric carcinoid, gastrointestinal, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (gist), germ cel tumor: extracranial, extragonadal, or ovarian, gestational, gestational trophoblastic tumor, glioma, glioma of the brain stem, hairy cel leukemia, hepatocelular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma, intraocular melanoma, islet cel carcinoma (endocrine pancreas), Kaposi sarcoma, kidney cancer (renal cel cancer), laryngeal cancer, leukaemias, lip and oral cavity cancer, liposarcoma, liver cancer (primary), lung cancer, lymphomas, macroglobulinemia, malignant fibrous histiocytoma of bone / osteosarcoma, meduloblastoma, melanoma, Merkel cel cancer, mesothelioma, metastatic, mouth cancer, multiple (cancer of the bone-marrow), multiple endocrine neoplasia syndrome, multiple myeloma / plasma cel neoplasm, mycosis fungoides, myelodysplastic syndromes, myelodysplastic / myeloproliferative diseases, myelogenous leukemia, myeloid leukemia, myeloma, myeloproliferative disorders, myxoma, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma neuroblastoma, non-Hodgkin lymphoma, non-smal cel, non-smal cel lung cancer, oligodendroglioma, oral cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma, ovarian cancer, ovarian epithelial cancer (surface epithelial-stromal tumor), ovarian germ cel tumor, ovarian low malignant potential tumor, pancreatic cancer, pancreatic cancer, islet cel, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary adenoma, plasma cel neoplasia / multiple myeloma, pleuropulmonary blastoma, primary central nervous system, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cel carcinoma, renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sezary syndrome, skin cancer (melanoma), skin cancer (non-melanoma), skin carcinoma, smal cel, smal cel lung cancer, smal intestine cancer, soft tissue, soft tissue sarcoma, squamous cel carcinoma, squamous neck cancer with occult primary, stomach cancer, supratentorial primitive neuroectodermal tumors, t-cel lymphoma, testicular cancer, throat cancer, thymoma, thymoma and thymic 139 43011.601_P18269-03carcinoma, thyroid cancer, transitional cel cancer, transitional cel cancer of the renal pelvis and ureter, trophoblastic tumor, unknown primary site, ureter and renal pelvis, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma, vulvar cancer, Waldenstrom, Waldenstrom macroglobulinemia, and Wilms tumor (kidney cancer).

[0228] In more particular embodiments, administering the compound of formula (I) or formula (I) in combination with the immunotherapy alters an immune cel microenvironment of a tumor associated with breast cancer, colorectal cancer, lung cancer, melanoma, pancreatic cancer, prostate cancer, or head and neck squamous cel carcinoma (HNSCC). In yet more particular embodiments, administering the compound of formula (I) or formula (I) in combination with an immunotherapy reduces a risk of an adverse event associated with the immunotherapy.

[0229] In certain embodiments, the immune checkpoint inhibitor targets an immune checkpoint receptor selected from the group consisting of CTLA-4, PD-1, PD-L1, PD-1 - PD-L1, PD-1 - PD- L2, interleukin-2 (IL-2), indoleamine 2,3 -dioxygenase (IDO), IL-10, transforming growth factor- P (TGFP), T cel immunoglobulin and mucin 3 (TIM3 or HAVCR2), Galectin 9 -TIM3, Phosphatidylserine - TIM3, lymphocyte activation gene 3 protein (LAG3), MHC class I - LAG3, 4- 1BB-4- IBB ligand, 0X40-0X40 ligand, GITR, GITR ligand - GITR, CD27, CD70-CD27, TNFRSF25, TNFRSF25-TL1A, CD40L, CD40-CD40 ligand, HVEM-LIGHT-LTA, HVEM, HVEM - BTLA, HVEM - CD 160, HVEM - LIGHT, HVEM-BTLA-CD160, CD80, CD80 - PDL- 1, PDL2 - CD80, CD244, CD48 - CD244, CD244, ICOS, ICOS-ICOS ligand, B7-H3, B7-H4, VISTA, TMIGD2, HHLA2-TMIGD2, Butyrophilins, including BTNL2, Siglec family, TIGIT and PVR family members, KIRs, ILTs and LIRs, NKG2D and NKG2A, MICA and MICB, CD244, CD28, CD86 - CD28, CD86 - CTLA, CD80 - CD28, CD39, CD73 Adenosine-CD39-CD73, CXCR4-CXCL12, Phosphatidylserine, TIM3, Phosphatidylserine - TIM3, SIRPA-CD47, VEGF, Neuropilin, CD160, CD30, and CD155; e.g., CTLA-4 or PD1 or PD-L1).

[0230] Immune checkpoint inhibitors include but not limited to Urelumab, PF-05082566, MEDI6469, TRX518, Varlilumab, CP-870893, Pembrolizumab (PD1), Nivolumab (PD1), Atezolizumab (formerly MPDL3280A) (PDL1), MEDI4736 (PD-L1), Avelumab (PD-L1), PDR001 (PD1), BMS-986016, MGA271, Lirilumab, IPH2201, Emactuzumab, INCB024360, Galunisertib, Ulocuplumab, BKT140, Bavituximab, CC-90002, Bevacizumab, and MNRP1685A, Durvalumab, Ipilimumab, REGN2810, BMS-936558 , SHR1210, KN035, IBI308, BGB-A317, BCD-100, JS001 and MGA271. 140 43011.601_P18269-03

[0231] In certain embodiments, the disease, disorder, or condition is associated with ocular neovascularization. In particular embodiments, the disease, disorder, or condition associated with ocular neovascularization is selected from diabetic macular edema, diabetic retinopathy, retinal vein occlusion, sickle cel retinopathy, retinopathy of prematurity, Norrie’s disease, Coat’s disease, corneal neovascularization, and age-related macular degeneration. In certain embodiments, administration of the compound of formula (I) prevents expression of VEGF and ANGPTL4.

[0232] In other embodiments, the method further comprises administering one or more additional therapeutic agents. In certain embodiments, the one or more additional therapeutic agents include an anti-cancer agent. In particular embodiments, the anti-cancer agent is selected from an angiogenesis inhibitor, an angiopoietin 2 inhibitor, a CD73 inhibitor, a cyclin dependent kinase inhibitor, an MAP kinase inhibitor, a mTOR inhibitor, a phosphatidylinositol 3-kinase inhibitor, a proteasome inhibitor, a protein phosphatase 2A activator, a serine / threonine kinase inhibitor, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a VEGF-A inhibitor, and a cytotoxic chemotherapy, including, but not limited to, 5-fluorouracil, adriamycin, carboplatin, cisplatin, doxorubicin, gemcitabine, idarubicin, and paclitaxel.

[0233] Other suitable anticancer agents include, but are not limited to, chemotherapeutic drug treatment, radiation, gene therapy, hormonal manipulation, immunotherapy and antisense oligonucleotide therapy. Chemotherapeutic agents include, but are not limited to alkylating agents, such as nitrogen mustards (for example, chlorambucil, chlormethine, cyclophosphamide, ifosfamide, and melphalan), nitrosoureas (for example, carmustine, fotemustine, lomustine, and streptozocin), platinum compounds (for example, carboplatin, cisplatin, oxaliplatin, and bbr3464), busulfan, dacarbazine, mechlorethamine, procarbazine, temozolomide, thiotepa, and uramustine; antimetabolites, such as folic acid (for example, methotrexate, pemetrexed, and raltitrexed), purine (for example, cladribine, clofarabine, fludarabine, mercaptopurine, and tioguanine), pyrimidine (for example, capecitabine), cytarabine, fluorouracil, and gemcitabine; plant alkaloids, such as podophylum (for example, etoposide, and teniposide), taxane (for example, docetaxel and paclitaxel), vinca (for example, vinblastine, vincristine, vindesine, and vinorelbine); cytotoxic / antitumor antibiotics, such as anthracycline family members (for example, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, and valrubicin), bleomycin, hydroxyurea, and mitomycin; topoisomerase inhibitors, such as topotecan and irinotecan; monoclonal antibodies, such as alemtuzumab, bevacizumab, cetuximab, gemtuzumab, rituximab, and tras tuzumab; 141 43011.601_P18269-03photosensitizers, such as aminolevulinic acid, methyl aminolevulinate, porfimer sodium, and verteporfm; and other agents, such as alitretinoin, altretamine, amsacrine, anagrelide, arsenic trioxide, asparaginase, bexarotene, bortezomib, celecoxib, denileukin diftitox, erlotinib, estramustine, gefitinib, hydroxycarbamide, imatinib, pentostatin, masoprocol, mitotane, pegaspaigase, and tretinoin.

[0234] In certain embodiments, the one or more additional therapeutic agents include an agent for treating a disease, disorder, or condition associated with ocular neovascularization. In particular embodiments, the one or more additional therapeutic agents for treating a disease, disorder, or condition associated with ocular neovascularization include an anti-VEGF agent. In more particular embodiments, the anti-VEGF agent is aflibercept, bevacizumab, faricimab, or ranubizumab.

[0235] In certain embodiments, the compound of formula (I) or formula (I) is administered prophylacticaly to prevent or reduce an incidence, recurrence, or progression of the disease, disorder, or condition.

[0236] In certain embodiments, the compound of formula(I) or formula (I): (a) directly binds the bHLH domain or the PAS-B subdomain of HIF-1α and HIF-2α; (b) disrupts dimerization with HIF-1β; (c) induces degradation of HIF-1α and HIF-2α; and (d) inhibits HIF target gene expression in one or more cels of the subject.

[0237] As used herein, the term “treating” can include reversing, aleviating, inhibiting the progression of, preventing, or reducing the likelihood of the disease, disorder, or condition to which such term applies, or one or more symptoms or manifestations of such disease, disorder, or condition. Preventing refers to causing a disease, disorder, condition, or symptom or manifestation of such, or worsening of the severity of such, not to occur. Accordingly, the presently disclosed compounds can be administered prophylacticaly to prevent or reduce the incidence or recurrence of the disease, disorder, or condition.

[0238] The “subject” treated by the presently disclosed methods in their many embodiments is desirably a human subject, although it is to be understood that the methods described herein are efective with respect to al vertebrate species, which are intended to be included in the term “subject.” Accordingly, a “subject” can include a human subject for medical purposes, such as for the treatment of an existing condition or disease or the prophylactic treatment for preventing the onset of a condition or disease, or an animal subject for medical, veterinary purposes, or 142 43011.601_P18269-03developmental purposes. Suitable animal subjects include mammals including, but not limited to, primates, e.g., humans, monkeys, apes, and the like; bovines, e.g., catle, oxen, and the like; ovines, e.g., sheep and the like; caprines, e.g., goats and the like; porcines, e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras, and the like; felines, including wild and domestic cats; canines, including dogs; lagomorphs, including rabbits, hares, and the like; and rodents, including mice, rats, and the like. An animal may be a transgenic animal. In some embodiments, the subject is a human including, but not limited to, fetal, neonatal, infant, juvenile, and adult subjects. Further, a “subject” can include a patient aflicted with or suspected of being afflicted with a condition or disease. Thus, the terms “subject” and “patient” are used interchangeably herein. The term “subject” also refers to an organism, tissue, cel, or colection of cels from a subject.

[0239] In general, a “therapeuticaly efective amount” of a therapeutic agent refers to the amount of the agent necessary to elicit the desired biological response. As wil be appreciated by those of ordinary skil in the art, the efective amount of an agent may vary depending on such factors as the desired biological endpoint, the agent to be delivered, the composition of the pharmaceutical composition, the target tissue or cel, and the like. In some embodiments, the term “therapeuticaly efective amount” refers to an amount suficient to reduce or ameliorate the severity, duration, progression, or onset of a disease, disorder, or condition, or one or more symptoms thereof; prevent the advancement of a disease, disorder, or condition, cause the regression of a disease, disorder, or condition; prevent the recurence, development, onset or progression of a symptom associated with a disease, disorder, or condition, or enhance or improve the prophylactic or therapeutic efect(s) of another therapy.

[0240] The term “combination” is used in its broadest sense and means that a subject is administered at least two agents, more particularly a compound disclosed herein and at least one other therapeutic agent. More particularly, the term “in combination” refers to the concomitant administration of two (or more) active agents for the treatment of a, e.g., single disease state. As used herein, the active agents may be combined and administered in a single dosage form, may be administered as separate dosage forms at the same time, or may be administered as separate dosage forms that are administered alternately or sequentialy on the same or separate days. In one embodiment of the presently disclosed subject mater, the active agents are combined and administered in a single dosage form. In another embodiment, the active agents are administered 143 43011.601_P18269-03in separate dosage forms (e.g., wherein it is desirable to vary the amount of one but not the other). The single dosage form may include additional active agents for the treatment of the disease state.

[0241] Further, the compounds disclosed herein can be administered alone or in combination with adjuvants that enhance stability of the compounds, alone or in combination with one or more therapeutic agents, facilitate administration of pharmaceutical compositions containing them in certain embodiments, provide increased dissolution or dispersion, increase inhibitory activity, provide adjunct therapy, and the like, including other active ingredients. Advantageously, such combination therapies utilize lower dosages of the conventional therapeutics, thus avoiding possible toxicity and adverse side efects incured when those agents are used as monotherapies.

[0242] The timing of administration of a compound disclosed herein and at least one additional therapeutic agent can be varied so long as the beneficial efects of the combination of these agents are achieved. Accordingly, the phrase “in combination with” refers to the administration of a compound described herein and at least one additional therapeutic agent either simultaneously, sequentialy, or a combination thereof. Therefore, a subject administered a combination of a compound described herein and at least one additional therapeutic agent can receive a compound and at least one additional therapeutic agent at the same time (i.e., simultaneously) or at diferent times (i.e., sequentialy, in either order, on the same day or on diferent days), so long as the efect of the combination of both agents is achieved in the subject.

[0243] When administered sequentialy, the agents can be administered within 1, 5, 10, 30, 60, 120, 180, 240 minutes or longer of one another. In other embodiments, agents administered sequentialy, can be administered within 1, 5, 10, 15, 20 or more days of one another. Where the compound described herein and at least one additional therapeutic agent are administered simultaneously, they can be administered to the subject as separate pharmaceutical compositions, each comprising either a compound or at least one additional therapeutic agent, or they can be administered to a subject as a single pharmaceutical composition comprising both agents.

[0244] When administered in combination, the efective concentration of each of the agents to elicit a particular biological response may be less than the effective concentration of each agent when administered alone, thereby alowing a reduction in the dose of one or more of the agents relative to the dose that would be needed if the agent was administered as a single agent. The efects of multiple agents may, but need not be, additive or synergistic. The agents may be administered multiple times. 144 43011.601_P18269-03

[0245] In some embodiments, when administered in combination, the two or more agents can have a synergistic effect. As used herein, the terms “synergy,” “synergistic,” “synergisticaly” and derivations thereof, such as in a “synergistic efect” or a “synergistic combination” or a “synergistic composition” refer to circumstances under which the biological activity of a combination of a compound described herein and at least one additional therapeutic agent is greater than the sum of the biological activities of the respective agents when administered individualy.

[0246] Synergy can be expressed in terms of a “Synergy Index (SI),” which generaly can be determined by the method described by F. C. Kul et al., Applied Microbiology 9, 538 (1961), from the ratio determined by:

[0247] Qa / QA + Qb / QB = Synergy Index (SI)

[0248] wherein:

[0249] QA is the concentration of a component A, acting alone, which produced an end point in relation to component A;

[0250] Qa is the concentration of component A, in a mixture, which produced an end point;

[0251] QB is the concentration of a component B, acting alone, which produced an end point in relation to component B; and

[0252] Qb is the concentration of component B, in a mixture, which produced an end point.

[0253] Generaly, when the sum of Qa / QA and Qb / QB is greater than one, antagonism is indicated. When the sum is equal to one, additivity is indicated. When the sum is less than one, synergism is demonstrated. The lower the SI, the greater the synergy shown by that particular mixture. Thus, a “synergistic combination” has an activity higher that what can be expected based on the observed activities of the individual components when used alone. Further, a “synergisticaly efective amount” of a component refers to the amount of the component necessary to elicit a synergistic efect in, for example, another therapeutic agent present in the composition.

[0254] FORMULATIONS AND METHODS OF ADMINISTRATION

[0255] Depending on the specific conditions being treated, the “agent(s)” may be formulated into liquid or solid dosage forms and administered systemicaly or localy. The agents may be delivered, for example, in a timed- or sustained-slow release form as is known to those skiled in the art. Techniques for formulation and administration may be found in Remington: The Science and Practice of Pharmacy (20th ed.) Lippincot, Wiliams & Wilkins (2000). Suitable routes may include oral, buccal, by inhalation spray, sublingual, rectal, transdermal, vaginal, transmucosal, 145 43011.601_P18269-03nasal or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedulary injections, as wel as intrathecal, direct intraventricular, intravenous, intra-articular, intra-sternal, intra-synovial, intra-hepatic, intralesional, intracranial, intraperitoneal, intranasal, or intraocular injections or other modes of delivery.

[0256] Generaly, routes for ocular delivery include topical, intravitreal, intraocular, juxtascleral, subconjunctival, intracameral, and retrobulbar. Such delivery can be efected via polymeric miceles, hydrogels, liposomes, niosomes, dendrimers, and cyclodextrins.

[0257] Topical delivery includes delivery via miceles, including β-cyclodextrin-based micelar systems, cubosomes, and proniosomal gels.

[0258] Intravitreal Injections / Implants involve delivery of a therapeutic agent into the vitreous that is close to the retina at the back of the eye. Such delivery methods include a single intravitreal injection of, for example, vitamin E / poly-lactic-co-glycolic acid microspheres or injection of polymer-free dexamethasone dimer implants.

[0259] Juxtascleral injections are used for treatment of some posterior part complaints that cannot be handled through conventional topical route. It is used, for example, for the treatment of cystoid macula edema, trauma, and diabetic-related conditions.

[0260] Subconjunctival injection is frequently used in cases of very low drug penetration into the anterior part of the eye after topical administration and can include PEGylated liposomes and PLGA nanoparticles.

[0261] Intracameral injections involve injection of the therapeutic agent directly into the anterior segment of the eyebal or in the vitreous cavity and include, for example, hydrogels functionalized with vinyl sulfone and thiol groups.

[0262] The retrobulbar route involves the injection of a needle through the eyelid and orbital fascia to deliver the therapeutic agent behind the globe into the retrobulbar space. For example, retrobulbar injection could be used to treat macular edema resulting from retinal vein occlusion.

[0263] Further, liquid dosage forms, including eye drops represent more than 95% of the marketed ocular products. They deliver the medication into the anterior part of the eye. Their advantages include easy administration and accepted stability. Their disadvantages, however, include low retention time (<5 min.), poor bioavailability, and serious side efect resulted from the frequent administration of high concentration. 146 43011.601_P18269-03

[0264] Several nanosystem platforms had been developed to solve their drawbacks including, for example, a mucoadhesive nanosystem including poly (D-L-lactide)-b-dextran, a micelar system, a hybridized vesicle solution, and a cyclodextrin nano-aggregate.

[0265] Eye suspensions can further include polymer systems and nanosuspensions. Eye emulsions can include nanoemulsions and microemulsions. Eye gels and ointments can include proniosomal gels, liquid crystaline gels, and cubosomal gels.

[0266] Further, ocular inserts can include nanofibers, microspheres, and nanostructured formulations. Therapeutic contact lens can include gold nanoparticles, nanoaggregates, and nanoparticles. It situ gels can include nanoparticles, solid lipid nanoparticle, nanostructured formulation, and niosomes.

[0267] In some embodiments, the delivery system can include a smart nano-micro platforms can change their mechanical, thermal, and / or optical properties in a manageable or expectable means, and they can achieve sensing triggering roles with stimuli-responsive features. Unlike conventional nanocariers, the smart nano-micro platforms can reveal precise reaction to exogenous (light, sound, and magnetic field) or endogenous (pH, reactive oxygen species, and biological molecules such as DNA and enzymes) factors resulting in accomplishing many functions, e.g., site-specific drug delivery, bio-imaging, and detection of bio-molecules.

[0268] For a review of curent ocular drug delivery systems, including nanotechnology-based systems, see Sadek Ahmed, Maha M. Amin, and Sinar Sayed, Ocular Drug Delivery: a Comprehensive Review, AAPS PharmSciTech (2023) 24:66; and Shiding Li, Liangbo Chen, and Yao Fu, Nanotechnology-based ocular drug delivery systems: recent advances and future prospects, Journal of Nanobiotechnology (2023) 21:232.

[0269] For injection, the agents of the disclosure may be formulated and diluted in aqueous solutions, such as in physiologicaly compatible bufers such as Hank’s solution, Ringer’s solution, or physiological saline bufer. For such transmucosal administration, penetrants appropriate to the barier to be permeated are used in the formulation. Such penetrants are generaly known in the art.

[0270] Use of pharmaceuticaly acceptable inert cariers to formulate the compounds herein disclosed for the practice of the disclosure into dosages suitable for systemic administration is within the scope of the disclosure. With proper choice of carrier and suitable manufacturing practice, the compositions of the present disclosure, in particular, those formulated as solutions, 147 43011.601_P18269-03may be administered parenteraly, such as by intravenous injection. The compounds can be formulated readily using pharmaceuticaly acceptable cariers wel known in the art into dosages suitable for oral administration. Such carriers enable the compounds of the disclosure to be formulated as tablets, pils, capsules, liquids, gels, syrups, sluries, suspensions, and the like, for oral ingestion by a subject (e.g., patient) to be treated.

[0271] For nasal or inhalation delivery, the agents of the disclosure also may be formulated by methods known to those of skil in the art, and may include, for example, but not limited to, examples of solubilizing, diluting, or dispersing substances, such as saline; preservatives, such as benzyl alcohol; absorption promoters; and fluorocarbons.

[0272] In particular embodiments, the compound disclosed herein is administered intranasaly in a form selected from the group consisting of a nasal spray, a nasal drop, a powder, a granule, a cachet, a tablet, an aerosol, a paste, a cream, a gel, an ointment, a salve, a foam, a paste, a lotion, a cream, an oil suspension, an emulsion, a solution, a patch, and a stick. As used herein, the term administrating via an "intranasal route" refers to administering by way of the nasal structures.

[0273] Pharmaceutical compositions suitable for use in the present disclosure include compositions wherein the active ingredients are contained in an efective amount to achieve its intended purpose. Determination of the efective amounts is wel within the capability of those skiled in the art, especialy in light of the detailed disclosure provided herein. Generaly, the compounds according to the disclosure are efective over a wide dosage range. For example, in the treatment of adult humans, dosages from 0.01 to 1000 mg, from 0.5 to 100 mg, from 1 to 50 mg per day, and from 5 to 40 mg per day are examples of dosages that may be used. A non-limiting dosage is 10 to 30 mg per day. The exact dosage wil depend upon the route of administration, the form in which the compound is administered, the subject to be treated, the body weight of the subject to be treated, the bioavailability of the compound(s), the adsorption, distribution, metabolism, and excretion (ADME) toxicity of the compound(s), and the preference and experience of the atending physician.

[0274] In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceuticaly acceptable cariers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceuticaly. The preparations formulated for oral administration may be in the form of tablets, dragees, capsules, or solutions. 148 43011.601_P18269-03

[0275] Pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipients, optionaly grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, filers such as sugars, including lactose, sucrose, mannitol, or sorbitol; celulose preparations, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl celulose, hydroxypropylmethyl-celulose, sodium carboxymethyl-celulose (CMC), and / or polyvinylpyrolidone (PVP: povidone). If desired, disintegrating agents may be added, such as the cross-linked polyvinylpyrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0276] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionaly contain gum arabic, talc, polyvinylpyrolidone, carbopol gel, polyethylene glycol (PEG), and / or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dye-stufs or pigments may be added to the tablets or dragee coatings for identification or to characterize diferent combinations of active compound doses.

[0277] Pharmaceutical preparations that can be used oraly include push-fit capsules made of gelatin, as wel as soft, sealed capsules made of gelatin, and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filer such as lactose, binders such as starches, and / or lubricants such as talc or magnesium stearate and, optionaly, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as faty oils, liquid parafin, or liquid polyethylene glycols (PEGs). In addition, stabilizers may be added.

[0278] Further, one of ordinary skil in the art wil recognize that the presently disclosed compounds, and pharmaceutical compositions thereof, include pharmaceuticaly acceptable salts. Pharmaceuticaly acceptable salts are generaly wel known to those of ordinary skil in the art, and include salts of active compounds that can be prepared with relatively nontoxic acids or bases, depending on the particular substituent moieties found on the compounds described herein. The parent form of the compound can difer from the various salt forms in certain physical properties, such as solubility, and the like.

[0279] When compounds of the present disclosure contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a suficient amount of the desired base, either neat or in a suitable inert solvent or by ion exchange, whereby 149 43011.601_P18269-03one basic counterion (base) in an ionic complex is substituted for another. Examples of pharmaceuticaly acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, magnesium, and the like.

[0280] When compounds of the present disclosure contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a suficient amount of the desired acid, either neat or in a suitable inert solvent or by ion exchange, whereby one acidic counterion (acid) in an ionic complex is substituted for another. Examples of pharmaceuticaly acceptable acid addition salts include those derived from inorganic acids, organic acids, and amino acids. See, for example, Berge et al, “Pharmaceutical Salts”, Journal of Pharmaceutical Science, 1977, 66, 1-19). Compounds containing both basic and acidic functionalities alow such compounds to be converted into either base or acid addition salts.

[0281] Accordingly, pharmaceuticaly acceptable salts suitable for use with the presently disclosed subject mater include, by way of example but not limitation, acetate, arginate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate, carbonate, monohydrogencarbonate, citrate, edetate, edisylate, estolate, esylate, fumarate, galactonate, gluceptate, gluconate, glutamate, glycolylarsanilate, hexylresorcinate, hydrabamine, hydriodic, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, isobutyrate, lactate, lactobionate, malate, maleate, malonate, mandelate, mesylate, methanesulfonate, mucate, napsylate, nitrate, pamoate (embonate), pantothenate, phosphate, phthalate, diphosphate, monohydrogen phosphate, dihydrogen phosphate, polygalacturonate, propionate, salicylate, stearate, subacetate, suberate, succinate, sulfate, monohydrogensulfate, tannate, tartrate, including (+)-tartrates, (-)-tartrates, and mixtures thereof including racemic mixtures, teoclate, p- toluenesulfonate and trifluoroacetate. Other pharmaceuticaly acceptable salts may be found in, for example, Remington: The Science and Practice of Pharmacy (20th ed.) Lippincot, Wiliams & Wilkins (2000).

[0282] DEFINITIONS

[0283] Unless otherwise noted, the chemical definitions provided immediately herein below are intended to comply with IUPAC. Compendium of Chemical Terminology, 2nd ed. (the "Gold Book"). Compiled by A. D. McNaught and A. Wilkinson. Blackwel Scientific Publications, Oxford (1997). 150 43011.601_P18269-03

[0284] The term “hydrocarbon” as used herein, refers to any chemical group comprising hydrogen and carbon. A hydrocarbon group may be substituted or unsubstituted. As would be known to one of ordinary skil in the art, al valencies must be satisfied in making any substitutions. The hydrocarbon may be unsaturated, saturated, branched, unbranched, cyclic, polycyclic, or heterocyclic.

[0285] The term “aliphatic” or “aliphatic group”, as used herein, means a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a monocyclic hydrocarbon or bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic (also referred to herein as “carbocyclyl”, “cycloaliphatic”, or “cycloalkyl”), that has a single point of atachment to the rest of the molecule. Unless otherwise specified, aliphatic groups contain 1-6 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-5 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-4 alipatic carbon atoms. In some embodiments, aliphatic groups contain 1-3 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-2 aliphatic carbon atoms. In some embodiments, “cycloaliphatic” (or “carbocyclyl” or “cycloalkyl”) refers to a monocyclic C3-C7 hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point of atachment to the rest of the molecule. Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl, or (cycloalkyl)alkenyl.

[0286] The term “alkane” refers to acyclic branched or unbranched hydrocarbons having the general formula CnH2n+2, and therefore consisting entirely of hydrogen atoms and saturated carbon atoms.

[0287] The term “alkyl” refers to a univalent group derived from an alkane by removal of a hydrogen atom from any carbon atom and having the chemical formula of -CnH2n+1. The groups derived by removal of a hydrogen atom from a terminal carbon atom of unbranched alkanes form a subclass of normal alkyl (n-alkyl) groups H(CH2)n. The groups RCH2, R2CH (R ≠ H), and R3C (R ≠ H) are primary, secondary and tertiary alkyl groups, respectively.

[0288] An alkyl can be a straight chain (i.e., unbranched) or branched acyclic hydrocarbon having the number of carbon atoms designated (i.e., C1-10 means one to ten carbons, including 1, 2, 3, 4, 151 43011.601_P18269-035, 6, 7, 8, 9, and 10 carbons). In particular embodiments, the term “alkyl” refers to C1-20 inclusive, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 carbons. In other embodiments, the alkyl can be a C1-C4 alkyl, including 1, 2, 3, and 4 carbons. In yet other embodiments, the alkyl can be a C1-C6 alkyl, including 1, 2, 3, 4, 5, and 6 carbons. In even yet other embodiments, the alkyl can be a C1-C8 alkyl, including 1, 2, 3, 4, 5, 6, 7, and 8 carbons.

[0289] “Lower alkyl” refers to an alkyl group having 1 to about 8 carbon atoms (i.e., a C1-8 alkyl), e.g., 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. “Higher alkyl” refers to an alkyl group having about 10 to about 20 carbon atoms, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. In certain embodiments, “alkyl” refers to straight-chain alkyls. In other embodiments, “alkyl” refers to branched alkyls. In certain other embodiments, “alkyl” refers to straight-chain and / or branched alkyls. “Branched” refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl or propyl, is atached to a linear alkyl chain.

[0290] Representative alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, sec-pentyl, isopentyl, neopentyl, n- hexyl, sec-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, and dodecyl.

[0291] Alkyl groups can optionaly be substituted (a “substituted alkyl”) with one or more substituents, which can be the same or diferent. Such substituent groups include, but are not limited to, alkyl, substituted alkyl, cycloalkyl, halogen, acyl, carboxyl, oxo, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, cyano, and mercapto.

[0292] The term “heteroalkyl,” by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain having from 1 to 20 carbon atoms or heteroatoms consisting of at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si and S, and wherein the nitrogen, phosphorus, and sulfur atoms may optionaly be oxidized and the nitrogen heteroatom may optionaly be quaternized. The heteroatom(s) O, N, P and S and Si may be placed at any interior position of the heteroalkyl group or at the position at which alkyl group is atached to the remainder of the molecule. Examples include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, - CH2-S-CH2-CH3, -CH2-CH2-S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, - CH2-CH=N-OCH3, -CH=CH-N(CH3)- CH3, O-CH3, -O-CH2-CH3, and -CN. Up to two or three heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3. 152 43011.601_P18269-03

[0293] The term “cycloalkane” refers to saturated monocyclic hydrocarbons (with or without side chains), e.g., cyclobutane. Unsaturated monocyclic hydrocarbons having one endocyclic double or one triple bond are caled cycloalkenes and cycloalkynes, respectively. Those having more than one such multiple bond are cycloalkadienes, cycloalkatrienes, and the like. The inclusive terms for any cyclic hydrocarbons having any number of such multiple bonds are cyclic olefins or cyclic acetylenes.

[0294] The term “cycloalkyl” refer to a univalent group derived from a cycloalkane by removal of a hydrogen atom from a ring carbon atom. Cycloalkyls can be a mono- or multicyclic ring system of about 3 to about 10 carbon atoms, e.g., 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. The cycloalkyl group also can be optionaly substituted with a substituent group provided hereinabove for alkyl groups. Representative monocyclic cycloalkyl rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. Multicyclic cycloalkyl rings include adamantyl, octahydronaphthyl, decalin, camphor, camphane, and noradamantyl, and fused ring systems, such as dihydro- and tetrahydronaphthalene, and the like.

[0295] The term “cycloalkylalkyl” as used herein, refers to a cycloalkyl group, which is atached to the parent molecular moiety through an alkylene moiety, also as defined above, e.g., a C1-20 alkylene moiety. Examples of cycloalkylalkyl groups include cyclopropylmethyl and cyclopentylethyl.

[0296] The terms “cycloheteroalkyl” and “heterocycloalkyl” (or more generaly “heterocyclic”) are used interchangeably and refer to an unsaturated ring system, such as a 3- to 10-member substituted or unsubstituted cycloalkyl ring system, including one or more heteroatoms, which can be the same or diferent, and are selected from the group consisting of nitrogen (N), oxygen (O), sulfur (S), phosphorus (P), and silicon (Si), in which the nitrogen, sulfur, and phosphorus heteroatoms may be oxidized and the nitrogen heteroatom may be quaternized. The cycloheteroalkyl ring can be optionaly fused to or otherwise atached to other cycloheteroalkyl rings and / or non-aromatic hydrocarbon rings. Representative cycloheteroalkyl ring systems include, but are not limited to pyrolidinyl, pyrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperidinyl, piperazinyl, indolinyl, quinuclidinyl, morpholinyl, thiomorpholinyl, thiadiazinanyl, tetrahydrofuranyl, and the like.

[0297] The terms “cycloalkylene” and “heterocycloalkylene” refer to the divalent derivatives of cycloalkyl and heterocycloalkyl, respectively. 153 43011.601_P18269-03

[0298] As used herein the terms “bicycloalkyl” and “bicycloheteroalkyl” refer to two cycloalkyl or cycloheteroalkyl groups that are bound to one another. Non-limiting examples include bicyclohexane and bipiperidine.

[0299] An “unsaturated hydrocarbon” has one or more double bonds or triple bonds. As used herein, the term “alkene” refers to an acyclic branched or unbranched hydrocarbons having one carbon–carbon double bond and the general formula CnH2n. Acyclic branched or unbranched hydrocarbons having more than one double bond are alkadienes, alkatrienes, and the like.

[0300] More particularly, the term “alkenyl” as used herein refers to a monovalent group derived from a C2-20 inclusive straight or branched hydrocarbon moiety having at least one carbon-carbon double bond by the removal of a single hydrogen molecule. Alkenyl groups include, but are not limited to, ethenyl (i.e., vinyl), 2-propenyl, butenyl, 1-methyl-2-buten-1-yl, pentenyl, 2- isopentenyl, hexenyl, octenyl, alenyl, butadienyl, crotyl (but-2-en-1-yl), 2-(butadienyl), 2,4- pentadienyl, 3-(l,4-pentadienyl), and the like, including higher homologs and isomers.

[0301] The term “cycloalkenyl” as used herein refers to a cyclic hydrocarbon containing at least one carbon-carbon double bond. Examples of cycloalkenyl groups include cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadiene, cyclohexenyl, 1,3-cyclohexadiene, cycloheptenyl, cycloheptatrienyl, and cyclooctenyl.

[0302] The term “alkyne” as used herein refers to an acyclic branched or unbranched hydrocarbons having a carbon-carbon triple bond and the general formula CnH2n-2, RC≡CR. Acyclic branched or unbranched hydrocarbons having more than one triple bond are known as alkadiynes, alkatriynes, and the like.

[0303] The term “alkynyl” as used herein refers to a monovalent group derived from a straight or branched C2-20 hydrocarbon of a designed number of carbon atoms containing at least one carbon- carbon triple bond. Examples of alkynyl groups include, but are not limited to, ethynyl, 2-propynyl (propargyl), 1-propynyl, pentynyl, hexynyl, and heptynyl groups, and the like.

[0304] As used herein, the term “alkylene” refers to an alkanediyl group having the free valencies on adjacent carbon atoms, e.g. –CH(CH3)CH2– propylene (systematicaly caled propane-1,2- diyl). More particularly, the term “alkylene” by itself or a part of another substituent refers to a straight or branched bivalent aliphatic hydrocarbon group derived from an alkyl group having from 1 to about 20 carbon atoms, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. The alkylene group can be straight, branched or cyclic. The alkylene group also can 154 43011.601_P18269-03be optionaly unsaturated and / or substituted with one or more “alkyl group substituents.” There can be optionaly inserted along the alkylene group one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms (also refered to herein as “alkylaminoalkyl”), wherein the nitrogen substituent is alkyl as previously described. Exemplary alkylene groups include methylene (–CH2– ); ethylene (–CH2–CH2–); propylene (–(CH2)3–); cyclohexylene (–C6H10–); –CH=CH–CH=CH–; –CH=CH–CH2–; -CH2CH2CH2CH2-, -CH2CH=CHCH2-, -CH2CsCCH2-, - CH2CH2CH(CH2CH2CH3)CH2-, -(CH2)q-N(R)-(CH2)r–, wherein each of q and r is independently an integer from 0 to about 20, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, and R is hydrogen or lower alkyl; methylenedioxyl (–O–CH2–O–); and ethylenedioxyl (-O-(CH2)2–O–). An alkylene group can have about 2 to about 3 carbon atoms and can further have 6-20 carbons. Typicaly, an alkyl (or alkylene) group wil have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being some embodiments of the present disclosure. A “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generaly having eight or fewer carbon atoms.

[0305] The term “heteroalkylene” by itself or as part of another substituent means a divalent group derived from heteroalkyl, as exemplified, but not limited by, -CH2-CH2-S-CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2-. For heteroalkylene groups, heteroatoms also can occupy either or both of the chain termini (e.g., alkyleneoxo, alkylenedioxo, alkyleneamino, alkylenediamino, and the like). Stil further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is writen. For example, the formula -C(O)OR’- represents both -C(O)OR’- and –R’OC(O)-.

[0306] The term “arene” refers to a monocyclic and polycyclic aromatic hydrocarbon

[0307] The term “aryl” refers to a group derived from arenes by removal of a hydrogen atom from a ring carbon atom. Groups similarly derived from heteroarenes are sometimes subsumed in this definition. An aryl group can include, for example, a single ring or multiple rings (such as from 2 to 3 rings), which are fused together or linked covalently.

[0308] The term “heteroaryl” refers to a group formed by removing one or more hydroxy groups from oxoacids that have the general structure RkE(=O)l(OH)m (l ≠ 0), and replacement analogues of such acyl groups. In organic chemistry an unspecified acyl group is commonly a carboxylic acyl group. 155 43011.601_P18269-03

[0309] The term “heteroaryl” refers to the class of heterocyclyl groups derived from heteroarenes by removal of a hydrogen atom from any ring atom. A “heteroaryl” group can include from one to four heteroatoms (in each separate ring in the case of multiple rings) selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionaly oxidized, and the nitrogen atom(s) are optionaly quaternized. A heteroaryl group can be atached to the remainder of the molecule through a carbon or heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrolyl, 2-pyrolyl, 3-pyrolyl, 3-pyrazolyl, 2- imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3- isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5- thiazolyl, 2-furyl, 3-furyl, 2- thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4- pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5- isoquinolyl, 2-quinoxalinyl, 5- quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituents for each of above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below. The terms “arylene” and “heteroarylene” refer to the divalent forms of aryl and heteroaryl, respectively.

[0310] For brevity, the term “aryl” when used in combination with other terms (e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroaryl rings as defined above. Thus, the terms “arylalkyl” and “heteroarylalkyl” are meant to include those groups in which an aryl or heteroaryl group is atached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl, furylmethyl, and the like) including those alkyl groups in which a carbon atom (e.g., a methylene group) has been replaced by, for example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(l- naphthyloxy)propyl, and the like). However, the term “haloaryl,” as used herein is meant to cover only aryls substituted with one or more halogens.

[0311] Where a heteroalkyl, heterocycloalkyl, or heteroaryl includes a specific number of members (e.g. “3 to 7 membered”), the term “member” refers to a carbon or heteroatom.

[0312] Each of above terms defined hereinabove (e.g. , “alkyl,” “heteroalkyl,” “cycloalkyl, and “heterocycloalkyl”, “alkenyl”, “alkynyl,” “aryl,” “heteroaryl,” as wel as their divalent derivatives) are meant to include both substituted and unsubstituted forms of the indicated group. Optional substituents for each type of group are provided below.

[0313] As used herein, the term “acyl” refers to a group formed by removing one or more hydroxy groups from oxoacids that have the general structure RkE(=O)l(OH)m (l ≠ 0), and replacement analogues of such acyl groups. In organic chemistry an unspecified acyl group is commonly a 156 43011.601_P18269-03carboxylic acyl group. For example, in some embodiments, the term acyl includes an organic acid group wherein the -OH of the carboxyl group has been replaced with another substituent and has the general formula RC(=O)-, wherein R is an alkyl, alkenyl, alkynyl, aryl, carbocylic, heterocyclic, or aromatic heterocyclic group as defined herein). As such, the term “acyl” specificaly includes arylacyl groups, such as a 2-(furan-2-yl)acetyl)- and a 2-phenylacetyl group. Specific examples of acyl groups include acetyl and benzoyl. Acyl groups also are intended to include amides, -RC(=O)NR’, esters, -RC(=O)OR’, ketones, -RC(=O)R’, and aldehydes, -RC(=O)H.

[0314] The terms “alkoxyl” or “alkoxy” are used interchangeably herein and refer to a saturated (i.e., alkyl–O–) or unsaturated (i.e., alkenyl–O– and alkynyl–O–) group atached to the parent molecular moiety through an oxygen atom, wherein the terms “alkyl,” “alkenyl,” and “alkynyl” are as previously described and can include C1-20 inclusive, linear, branched, or cyclic, saturated or unsaturated oxo-hydrocarbon chains, including, for example, methoxyl, ethoxyl, propoxyl, isopropoxyl, n-butoxyl, sec-butoxyl, tert-butoxyl, and n-pentoxyl, neopentoxyl, n-hexoxyl, and the like.

[0315] The term “alkoxyalkyl” as used herein refers to an alkyl-O-alkyl ether, for example, a methoxyethyl or an ethoxymethyl group.

[0316] “Aryloxyl” refers to an aryl-O- group wherein the aryl group is as previously described, including a substituted aryl. The term “aryloxyl” as used herein can refer to phenyloxyl or hexyloxyl, and alkyl, substituted alkyl, halo, or alkoxyl substituted phenyloxyl or hexyloxyl.

[0317] “Aralkyl” refers to an aryl-alkyl-group wherein aryl and alkyl are as previously described, and included substituted aryl and substituted alkyl. Exemplary aralkyl groups include benzyl, phenylethyl, and naphthylmethyl.

[0318] “Aralkyloxyl” refers to an aralkyl-O– group wherein the aralkyl group is as previously described. An exemplary aralkyloxyl group is benzyloxyl, i.e., C6H5-CH2-O-. An aralkyloxyl group can optionaly be substituted.

[0319] “Alkoxycarbonyl” refers to an alkyl-O-C(=O)– group. Exemplary alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl, butyloxycarbonyl, and tert-butyloxycarbonyl.

[0320] “Aryloxycarbonyl” refers to an aryl-O-C(=O)– group. Exemplary aryloxycarbonyl groups include phenoxy- and naphthoxy-carbonyl. 157 43011.601_P18269-03

[0321] “Aralkoxycarbonyl” refers to an aralkyl-O-C(=O)– group. An exemplary aralkoxycarbonyl group is benzyloxycarbonyl.

[0322] The term “acyloxyl” refers to an oxygen-centered radicals consisting of an acyl radical bonded to an oxygen atom, e.g., an acyl-O- group wherein acyl is as previously described.

[0323] The term “amine” refers to a compound formaly derived from ammonia by replacing one, two or three hydrogen atoms by hydrocarbyl groups, and having the general structures RNH2 (primary amines), R2NH (secondary amines), R3N (tertiary amines). In some embodiments, the term amino refers to the –NH2 group. More generaly, the amino group is -NR'R”, wherein R' and R” are typicaly selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0324] The terms “acylamino” and “alkylamino” refer to specific N-substituted organic radicals with acyl and alkyl substituent groups respectively.

[0325] An “aminoalkyl” as used herein refers to an amino group covalently bound to an alkylene linker. More particularly, the terms alkylamino, dialkylamino, and trialkylamino as used herein refer to one, two, or three, respectively, alkyl groups, as previously defined, atached to the parent molecular moiety through a nitrogen atom. The term alkylamino refers to a group having the structure –NHR’ wherein R’ is an alkyl group, as previously defined; whereas the term dialkylamino refers to a group having the structure –NR’R”, wherein R’ and R” are each independently selected from the group consisting of alkyl groups. The term trialkylamino refers to a group having the structure –NR’R”R”’, wherein R’, R”, and R’” are each independently selected from the group consisting of alkyl groups. Additionaly, R’, R”, and / or R’” taken together may optionaly be –(CH2)k– where k is an integer from 2 to 6. Examples include, but are not limited to, methylamino, dimethylamino, ethylamino, diethylamino, diethylaminocarbonyl, methylethylamino, isopropylamino, piperidino, trimethylamino, and propylamino.

[0326] The terms alkylthioether and thioalkoxyl refer to a saturated (i.e., alkyl–S–) or unsaturated (i.e., alkenyl–S– and alkynyl–S–) group atached to the parent molecular moiety through a sulfur atom. Examples of thioalkoxyl moieties include, but are not limited to, methylthio, ethylthio, propylthio, isopropylthio, n-butylthio, and the like.

[0327] “Acylamino” refers to an acyl-NH– group wherein acyl is as previously described. “Aroylamino” refers to an aroyl-NH– group wherein aroyl is as previously described. 158 43011.601_P18269-03

[0328] The term “carbonyl” refers to a compound containing the carbonyl group, -C(=O)-. The term is commonly used in the restricted sense of aldehydes (R-C(=O)H) and ketones, although it actualy includes carboxylic acids and derivatives.

[0329] The term “carboxylic acid” refers to an oxoacids having the structure RC(=O)OH. The term is used as a sufix in systematic name formation to denote the –C(=O)OH group including its carbon atom. In some embodiments, the term “carboxyl” refers to the –COOH group. Such groups also are refered to herein as a “carboxylic acid” moiety.

[0330] “Carbamoyl” refers to an amide group of the formula –C(=O)NH2.

[0331] “Alkylcarbamoyl” refers to a R’RN–C(=O)– group wherein one of R and R’ is hydrogen and the other of R and R’ is alkyl and / or substituted alkyl as previously described.

[0332] “Dialkylcarbamoyl” refers to a R’RN–C(=O)– group wherein each of R and R’ is independently alkyl and / or substituted alkyl as previously described.

[0333] The term carbonyldioxyl, as used herein, refers to a carbonate group of the formula -O- C(=O)-OR.

[0334] The term “cyano” refers to the -C≡N group.

[0335] The terms “halo,” “halide,” or “halogen” as used herein refer to fluoro, chloro, bromo, and iodo groups. Additionaly, terms such as “haloalkyl,” are meant to include monohaloalkyl and polyhaloalkyl. For example, the term “halo(C1-4)alkyl” is mean to include, but not be limited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

[0336] The term “hydroxyl” refers to the –OH group.

[0337] The term “hydroxyalkyl” refers to an alkyl group substituted with an –OH group.

[0338] The term “mercapto” refers to the –SH group.

[0339] The term “oxo compound” refers to a compounds containing an oxygen atom, =O, doubly bonded to carbon or another element. The term thus embraces aldehydes, carboxylic acids, ketones, sulfonic acids, amides and esters. Oxo used as an adjective (and thus separated by a space) modifying another class of compound, as in oxo carboxylic acids, indicates the presence of an oxo substituent at any position. To indicate a double-bonded oxygen that is part of a ketonic structure, the term keto is sometimes used as a prefix, but such use has been abandoned by IUPAC for naming specific compounds. A traditional use of keto is for indicating oxidation of CHOH to C=O in a parent compound that contains OH groups, such as carbohydrates, e.g.3-ketoglucose. In some 159 43011.601_P18269-03embodiments, the term “oxo” as used herein means an oxygen atom that is double bonded to a carbon atom or to another element.

[0340] The term “nitro” refers to the –NO2 group.

[0341] The term “thio” refers to replacement of an oxygen by a sulfur, e.g., PhC(=S)NH2, thiobenzamide.

[0342] The term “thiol” refers to a compounds having the structure RSH (R ≠ H), e.g., MeCH2SH ethanethiol. A thiol also is known by the term “mercaptan.”

[0343] The term “thiohydroxyl” or “thiol,” as used herein, refers to a group of the formula –SH.

[0344] The term “sulfate” refers to the –SO4 group.

[0345] The term “sulfide” refers to a compound having the structure RSR (R ≠ H) and also are refered to as “thioethers.”

[0346] The term “sulfone” refers to a compound having the structure, RS(=O)2R (R ≠ H), e.g., C2H5S(=O)2CH3 ethyl methyl sulfone.

[0347] The term “sulfoxide” refers to a compound having the structure R2S=O (R ≠ H), e.g., Ph2S=O diphenyl sulfoxide.

[0348] The term “ureido” refers to a urea group of the formula –NH—CO—NH2.

[0349] One of ordinary skil in the art would recognize that a structure represented generaly by, for example, the formula: used herein refers to a ring structure, for example,carbon, a 6-carbon, a 7-carbon, and the like, aliphatic and / or aromatic cyclic compound, including a saturated ring structure, a partialy saturated ring structure, and an unsaturated ring structure, comprising a substituent R group, wherein the R group can be present or absent, and when present, one or more R groups can each be substituted on one or more available carbon atoms of the ring structure. The presence or absence of the R group and number of R groups is determined by the value of the variable “n,” which is an integer generaly having a value ranging from 0 to the number of carbon atoms on the ring available for substitution. Each R group, if more than one, is substituted on an available carbon of the ring structure rather than on another R group. For example, the structure above where n is 0 to 2 would comprise compound groups including, but not limited to: 160 43011.601_P18269-03ke. cture indicates that the bond can be either present or absent in the ring. That is, a dashed line representing a bond in a cyclic ring structure indicates that the ring structure is selected from the group consisting of a saturated ring structure, a partialy saturated ring structure, and an unsaturated ring structure.

[0353] The symbol ( ) denotes the point of atachment of a moiety to the remainder of the molecule.

[0354] When a named atom of an aromatic ring or a heterocyclic aromatic ring is defined as being “absent,” the named atom is replaced by a direct bond.

[0355] Throughout the specification and claims, a given chemical formula or name shal encompass al tautomers, congeners, and optical- and stereoisomers, as wel as racemic mixtures where such isomers and mixtures exist.

[0356] Certain compounds of the present disclosure may possess asymmetric carbon atoms (optical or chiral centers) or double bonds; the enantiomers, racemates, diastereomers, tautomers, geometric isomers, stereoisometric forms that may be defined, in terms of absolute stereochemistry, as (R)-or (S)- or, as D- or L- for amino acids, and individual isomers are encompassed within the scope of the present disclosure. The compounds of the present disclosure do not include those which are known in art to be too unstable to synthesize and / or isolate. The present disclosure is meant to include compounds in racemic, scalemic, and opticaly pure forms. Opticaly active (R)- and (S)-, or D- and L-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. When the compounds described herein contain olefenic bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers.

[0357] Unless otherwise stated, structures depicted herein are also meant to include al stereochemical forms of the structure; i.e., the R and S configurations for each asymmetric center. 161 43011.601_P18269-03Therefore, single stereochemical isomers as wel as enantiomeric and diastereomeric mixtures of the present compounds are within the scope of the disclosure.

[0358] As used herein, the term “congener” refers to one of two or more substances related to each other by origin, structure, or function.

[0359] The term “enantiomer” refers to one of a pair of molecular entities which are miror images of each other and non-superposable.

[0360] The term “stereoisomer” refers to an isomer that possess identical constitution, but which difer in the arangement of their atoms in space.

[0361] The term “racemate” refers to an equimolar mixture of a pair of enantiomers. It does not exhibit optical activity. The chemical name or formula of a racemate is distinguished from those of the enantiomers by the prefix (±)- or rac- (or racem-) or by the symbols RS and SR.

[0362] The term “diastereoisomerism” refers to stereoisomerism other than enantiomerism. Diastereoisomers (or diastereomers) are stereoisomers not related as miror images. Diastereoisomers are characterized by diferences in physical properties, and by some diferences in chemical behavior towards achiral as wel as chiral reagents.

[0363] It wil be apparent to one skiled in the art that certain compounds of this disclosure may exist in tautomeric forms, al such tautomeric forms of the compounds being within the scope of the disclosure. The term “tautomer,” as used herein, refers to one of two or more structural isomers which exist in equilibrium and which are readily converted from one isomeric form to another.

[0364] The term “about,” when used in connection with one or more numbers or numerical ranges, should be understood to refer to al such numbers, including al numbers in a range and modifies that range by extending the boundaries slightly above and slightly below the numerical values set forth by, for example, in some embodiments, + / -20%, + / -15%, + / -10%, + / -5%, + / -4%, + / -3%, + / - 2%, and + / -1%. The recitation of numerical ranges by endpoints includes al numbers, e.g., whole integers, including fractions thereof, subsumed within that range (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as wel as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) and any range within that range.

[0365] The phrase “in one embodiment” or “in some embodiments” as used herein does not necessarily refer to the same embodiment, though it may. Furthermore, the phrase “in another embodiment” as used herein does not necessarily refer to a diferent embodiment, although it may. 162 43011.601_P18269-03Thus, as described below, various embodiments of the invention may be readily combined, without departing from the scope or spirit of the invention.

[0366] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references, i.e., “one or more,” unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentialy of,” the embodiments or elements presented herein, whether explicitly set forth or not. Likewise, the term “include” and its grammatical variants are intended to be non- limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items. EXAMPLES

[0367] The folowing Examples have been included to provide guidance to one of ordinary skil in the art for practicing representative embodiments of the presently disclosed subject mater. In light of the present disclosure and the general level of skil in the art, those of skil can appreciate that the folowing Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject mater. The synthetic descriptions and specific examples that folow are only intended for the purposes of ilustration and are not to be construed as limiting in any manner to make compounds of the disclosure by other methods. EXAMPLE 1

[0368] Smal Molecules Targeting the bHLH and PAS Domains of Hypoxia-Inducible Factors for Cancer Therapy

[0369] Overview

[0370] Hypoxia-inducible factors (HIFs) function as master regulators of cancer progression by regulating angiogenesis, cancer stem cel specification, epithelial-mesenchymal transition, immune evasion, tissue invasion and metastasis. The HIF-2 antagonist belzutifan has been approved for treatment of renal cel carcinoma. In many cancers, both HIF-1 and HIF-2 drive progression, but no drugs that bind to both HIFs have been developed. We utilized computer-aided drug discovery and cel-based reporter assays to identify HIF-1 / 2 inhibitors. These compounds bind directly to HIF-1α and HIF-2α, disrupt dimerization with HIF-1β and trigger proteasomal 163 43011.601_P18269-03degradation, thereby inhibiting HIF transcriptional activity. These dual HIF-1 / 2 inhibitors blocked growth and vascularization of breast, colorectal, head / neck, lung, melanoma, and prostate tumors as monotherapy and increased responses to anti-CTLA4 or anti-PD-1 antibody therapy, with an aggregate complete response rate of over 50%, through broad reprogramming of the tumor immune cel microenvironment. When tested head-to-head versus the HIF-2-selective inhibitor PT2385, our dual HIF-1 / 2 inhibitor 1.21S9N showed superior anti-tumor activity in a mouse model of colorectal cancer. PT2385 caused breathing abnormalities whereas 1.21S9N did not.1.21S9N, 3.2.16, and related compounds are the first dual HIF-1 / 2 inhibitors that bind to defined domains of both proteins, show activity against a wide range of cancers, are oraly bioavailable, and dramaticaly improve responses to immune checkpoint blockade.

[0371] Hypoxia-inducible factors HIF-1 and HIF-2 play critical roles in cancer progression. We have developed the first dual HIF-1 / 2 inhibitors that bind to defined domains of both proteins, show activity against a wide range of cancers, are oraly bioavailable, and improve responses to immune checkpoint blockade.

[0372] This Example describes the development of smal-molecule dual HIF-1 / HIF-2 inhibitors that bind directly to HIF-α subunits, block dimerization with HIF-1β, and cause HIF-α degradation. The presently disclosed compounds inhibited HIF target gene expression in cancer cel lines at submicromolar concentrations, inhibited tumor growth as monotherapy and, in combination with ICB, markedly increased rates of tumor eradication in mouse models of melanoma, breast, colorectal, lung and prostate cancer.

[0373] Background

[0374] Hypoxia, defined as decreased O2 availability, is a common feature of many pathological conditions, including cancer, ischemic cardiovascular disease, and inflammatory disorders (Eltzschig and Carmeliet, 2011; Giaccia et al., 2003; Semenza, 2012a; Yuan et al., 2024). In solid tumors, dysregulated cancer cel proliferation and recruitment of immune cels, coupled with the generation of blood vessels that are structuraly and functionaly abnormal, leads to a mismatch of O2 supply and demand, including regions with severe intratumoral hypoxia (Bertout et al., 2008; Vaupel et al., 2007; Wicks and Semenza, 2022; Wilson and Hay, 2011). Direct measurements by Eppendorf microelectrode revealed that in normal breast tissue the median pO2 was 65 mmHg and no value less than 12.5 mmHg was recorded, whereas in advanced breast cancers, the median pO2 was 10 mmHg (Vaupel et al., 2007). In prostate cancer, a median pO2 value of 2.4 mmHg was 164 43011.601_P18269-03reported (Movsas et al., 2002). The presence of intratumoral hypoxia is independent of tumor grade, stage, or size and is a predictor of metastasis, treatment failure, and patient mortality (Vaupel et al., 2007).

[0375] In response to hypoxia, cels in most metazoans activate a transcriptional pathway mediated by hypoxia-inducible factors (HIFs), which play a crucial role in adaptation to low O2 levels (Semenza, 2012a; Yuan et al., 2024). HIFs are heterodimeric transcription factors composed of an O2-sensitive HIF-α subunit (HIF-1α, HIF-2α, or HIF-3α) and a constitutively expressed HIF-1β subunit (Wang et al., 1995; Yuan et al., 2024). The amino-terminal half of both subunits consists of basic-helix-loop-helix (bHLH) and Per-Arnt-Sim (PAS) domains that are required for dimerization and DNA binding (Jiang et al., 1996; Wang et al., 1995). The carboxy-terminal half of the HIF-α subunits consists of O2-dependent degradation and transactivation domains (Jiang et al., 1997). Under normoxic conditions, HIF-α subunits are subject to O2-dependent hydroxylation and binding of the VHL protein, which targets HIF-α subunits for ubiquitination and proteasomal degradation (Epstein et al., 2001). When O2 levels decrease, hydroxylation is inhibited, HIF-α subunits dimerize with HIF-1β, bind to hypoxia response elements (HREs), and activate transcription of target genes (Yuan et al., 2024). Oncogene gain-of-function and tumor suppressor loss-of-function mutations increase HIF activity in an O2-independent manner (Semenza, 2010).

[0376] Expression of HIF target genes (Winter et al., 2007) and expression of HIF-1α or HIF-2α protein (Wicks and Semenza, 2022) in tumor biopsies are associated with patient mortality in many cancers, reflecting the role of HIFs in directing tumor vascularization, metabolic reprogramming, epithelial-mesenchymal transition, cel motility, extracelular matrix remodeling, cancer stem cel specification, immune evasion, invasion, metastasis and treatment failure (Noman et al., 2019; Rankin et al., 2016; Sitkovsky et al., 2014; Wicks and Semenza, 2022). Although HIF-1α and HIF- 2α are both O2-regulated and show high sequence similarity in the bHLH and PAS domains, they are expressed in diferent tumor niches and have non-overlapping roles in various human cancer and associated stromal cels, with HIF-1α mediating acute hypoxic responses and glycolysis, and HIF-2α driving chronic hypoxia responses and MYC-mediated proliferation (Cowman and Koh, 2022; Gordan et al., 2007; Keith et al., 2011; Wigerup et al., 2016; Yuan et al., 2024). Thus, dual HIF-1 / 2 inhibition strategies are needed to achieve efective and sustained anti-tumor responses.

[0377] The interaction of immune checkpoint receptors CTLA-4 and PD-1 on T cels and natural kiler (NK) cels with their cognate ligands CD80 / 86 and PD-L1, respectively, on antigen- 165 43011.601_P18269-03presenting cels and cancer cels induces T and NK cel exhaustion or apoptosis, and immune checkpoint blockade (ICB) through the administration of antibodies against CTLA-4 (a-CTLA4), PD-1 (α-PD-1) and PD-L1 has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of many cancers (Sharma et al., 2021; Topalian et al., 2015). Although ICB can lead to tumor eradication, it is inefective in the majority of patients, either because T and NK cels have been excluded from the tumor or are present but do not respond to ICB because the cancer has employed other mechanisms of immune evasion (Crespo et al., 2013; Zou, 2005). Hypoxia alters the tumor immune-cel microenvironment (TIME) to favor immunosuppression over anti- tumor immunity (Chouaib et al., 2012; Noman et al., 2019; Semenza, 2021; Sitkovsky et al., 2014). HIF-1 mediates increased expression of PD-L1, which binds to PD-1 on T and NK cels to induce exhaustion or apoptosis (Barsoum et al., 2014; Noman et al., 2014), and CD73, an extracelular enzyme that generates adenosine, which also binds to cognate receptors on T and NK cels to induce exhaustion or apoptosis (Sitkovsky et al., 2014; Synnestvedt et al., 2002). Cancers expressing both CD73 and PD-L1 are resistant to therapies targeting either one.

[0378] Belzutifan (PT2977) binds selectively to the PAS-B subdomain of HIF-2α to block dimerization with HIF-1β and is approved by the U.S. FDA for the treatment of renal cel carcinoma (RCC) (Falah et al., 2024; Jonasch et al., 2021). However, it is unknown whether selective inhibition of HIF-2 wil benefit patients with other types of cancer. Given the distinct roles of HIF-1 and HIF-2 in cancer progression, dual inhibition presents a promising therapeutic strategy, particularly for cancer types with a known propensity for intratumoral hypoxia and / or resistance to conventional therapy (Semenza, 2012b; Wicks and Semenza, 2022; Wilson and Hay, 2011; Yuan et al., 2024). Several HIF inhibitors, including YC-1 (Yeo et al., 2003), echinomycin (Kong et al., 2005), PX-478 (Koh et al., 2008), IDF-1174 (Shen et al., 2022), and 32-134D (Salman et al., 2022), have anti-tumor activity as monotherapy and potentiate the effect of ICB in mouse models (Bailey et al., 2022; Luo et al., 2022; Ma et al., 2022; Salman et al., 2022; Shen et al., 2022). However, unlike belzutifan and the related compound PT2385 (Walace et al., 2016), none of these other inhibitors have been shown to bind directly to HIFs. Here, we describe smal- molecule dual HIF-1 / 2 inhibitors that bind directly to defined domains of HIF-α subunits, block dimerization with HIF-1β, and cause HIF-α degradation.

[0379] Results

[0380] Identification of candidate ligand binding sites in HIF-2α 166 43011.601_P18269-03

[0381] Computer-aided drug discovery using the crystal structure of the HIF-2α:HIF-1β complex (PDB: 4ZP4; Wu et al., 2015) and the site identification by ligand competitive saturation (SILCS) approach (Guvench and MacKerel, 2009) was performed. SILCS simulations produce three- dimensional distributions of grid free energies (GFEs) of functional groups throughout the protein termed FragMaps (MacKerel et al., 2020), which were used to identify three potential ligand binding sites in the bHLH domain (site 1), PAS-A subdomain (site 2), and PAS-B subdomain (site 3) of HIF-2α (FIG.1A-FIG.1C). FragMaps were utilized to generate pharmacophore features, which identify the types of functional groups and spatial relationships between those features that drug-like molecules should possess in order to bind at each site (FIG.1A). Virtual database screening was performed by selecting pharmacophore hypotheses that each contained a subset of 3 or 4 out of the total number of features at each site in various spatial relationships (see Materials and Methods). These pharmacophore hypotheses were individualy screened against an in-house database containing 768,466 compounds with ranking of compounds performed using the SILCS Monte Carlo approach. The top 100 candidate ligands at each site with the lowest ligand GFE values were chosen for further evaluation.

[0382] Identification of compounds that inhibit HIF transcriptional activity

[0383] Of the 300 candidate compounds, 293 were commercialy available and screened for inhibition of HIF-1 transcriptional activity in Hep3B-c1 cels, which are stably transfected with: HIF-1-dependent reporter plasmid p2.1, in which firefly luciferase (FLuc) coding sequences are downstream of the ENO1 HRE and a basal SV40 promoter; and control reporter plasmid pSV-RL, in which Renila luciferase (RLuc) coding sequences are downstream of the SV40 promoter only, such that the Fluc / RLuc ratio served as a measure of HIF-1 transcriptional activity (Salman et al., 2022). Alternatively, we transfected Hep3B cels with: a HIF-2α expression vector; control reporter pSV-RL; and pEPO-ProEn-FL, a reporter containing Fluc coding sequences under the control of EPO promoter and enhancer sequences, such that the Fluc / RLuc ratio served as a measure of HIF-2 activity (Dioum et al., 2009). Cels were exposed to compounds and incubated at 20% or 1% O2 for 24 hours. Compounds that decreased the Fluc / RLuc ratio by greater than 50% at low micromolar concentrations were tested for their effect on the hypoxia-induced expression of endogenous CA9 and EPO mRNA, which is mediated by HIF-1 and HIF-2, respectively.

[0384] Site 1 ligand SS1.21 (4,4’-(propane-1,3-diyl)bis(N-phenylpiperidine-1-carbothio-amide), which was identified in both the HIF-1 and HIF-2 reporter assays, and site 3 ligand SS3.2 (3-(6- 167 43011.601_P18269-03(2-phenoxyethyl)piperazin-1-yl)pyrimidin-4-yl)cyclobutan-1-ol), which was identified in the HIF- 2 reporter assay, were found to inhibit endogenous HIF target gene expression in Hep3B cels (FIG.1B-FIG.1F). In agreement with the reporter assays, SS3.2 showed a lower IC50 for the HIF- 2 target EPO (0.3 μM) as compared to the HIF-1 target CA9 (1.6 μM), whereas for SS1.21, the IC50 for EPO was 0.6 μM and for CA9 was 1.2 μM (FIG.1E and FIG.1F). With respect to EPO gene expression, the inhibitory activity of SS3.2 (0.3 μM) was comparable to that of the selective HIF-2 inhibitors PT2385 (0.3 μM) and PT2977 (0.2 μM), whereas the later compounds did not inhibit CA9 expression (FIG.1E and FIG.1F). None of the compounds had any effect on RPL13A mRNA expression, which is neither hypoxia-induced nor HIF-regulated (FIG.2A).

[0385] SS1.21 has a high estimated log P = 6.1, indicative of hydrophobicity and poor aqueous solubility. We generated 83 derivatives with decreased log P and tested their HIF inhibitory activity, leading to identification of 1.21S9N (4,4’-(propane-1,3-diyl)bis(N-(pyridine-4- ylmethyl)piperidine-1-carbothioamide) (FIG.1D-FIG.1F). We also screened 346 compounds, which had structural similarity to SS1.21 or SS3.2 and were predicted to maintain interaction with the HIF-2α:HIF-1β complex, in the cel-based reporter assay and identified 3.2.16 (cis-3-(6-(2- (2-ethylphenoxy)ethyl)(methyl)amino) pyrimidin-4-yl) cyclobutan-1-ol) (FIG.1D-FIG.1F).

[0386] Identification of mechanism and activity in many cancer cel types

[0387] Treatment of Hep3B cels with SS1.21 (FIG.3A) or SS3.2 (FIG.3B) blocked hypoxia- induced HIF-1α and HIF-2α protein accumulation. HIF-1β was constitutively expressed in al conditions (FIG.3A and FIG.3B). Treatment with SS1.21 or SS3.2 had no significant efect on HIF-1α or HIF-2α mRNA levels (FIG.3B) or β-actin protein levels (FIG.3C). SS1.21 or SS3.2 blocked accumulation of HIF-1α-DM, a hydroxylation-resistant form of HIF-1α in which Pro-402 and Pro-564 were mutated to alanine (FIG.3C). SS1.21 (FIG.3D and FIG.3E) or SS3.2 (FIG.3F and FIG.3G) also inhibited HIF-1α and HIF-2α expression in cels treated with dimethyloxalylglycine, which blocks O2-dependent prolyl hydroxylation. MG132, which inhibits proteasome-dependent degradation, rescued HIF-1α expression in SS1.21- (FIG.3H) or SS3.2- (FIG.3I) treated cels, whereas bafilomycin, an inhibitor of lysosome-dependent degradation, did not (FIG.3H and FIG.3I). Treatment with TAK243 to block protein ubiquitination also rescued HIF-1α expression in cels treated with SS1.21 (FIG.3J) or SS3.2 (FIG.3K).

[0388] Without wishing to be bound to any one particular theory, it is thought that binding of SS1.21 or SS3.2 to site 1 or site 3, respectively, might inhibit heterodimerization of HIF-1α and 168 43011.601_P18269-03HIF-1β prior to protein degradation. To test this hypothesis, we performed immunoprecipitation assays using lysates prepared from cels treated with SS1.21 or SS3.2 in the presence of MG132. HIF-1α was detected in lysates of MG132-treated cels exposed to 20% or 1% O2, either in the presence or absence of SS1.21 (FIG.3L and FIG.3D) or SS3.2 (FIG.3M and FIG.3D); however, HIF-1α co-immunoprecipitated with HIF-1β only in the absence of SS1.21 or SS3.2.

[0389] Without wishing to be bound to any one particular theory, it was thought that if binding of SS1.21 or SS3.2 to site 1 or site 3, respectively, triggers HIF-1α degradation, then deletion of the bHLH or PAS domain, respectively, should render the protein resistant to degradation. Hep3B cels were transfected with an expression vector encoding HIF-1α that was either ful-length (FL) or lacked residues 33-71 of the bHLH domain (ΔbHLH), which encompass site 1, or residues 85- 298 of the PAS domain (ΔPAS), which encompass site 3 (FIG.3N). SS1.21 induced the degradation of HIF-1α(FL) and HIF-1α(ΔPAS), but not HIF-1α(ΔbHLH), whereas SS3.2 induced the degradation of HIF-1α(FL) and HIF-1α(ΔbHLH), but not HIF-1α(ΔPAS) (FIG.3O). The data indicate that SS1.21 and SS3.2 bind to HIF-α bHLH and PAS domains, respectively, which blocks dimerization with HIF-1β and causes degradation of HIF-α subunits in a hydroxylation- independent and ubiquitin / proteasome-dependent manner.

[0390] Next, we analyzed the effect of each HIF inhibitor (HIFi) on hypoxia-induced HIF target gene expression in human and mouse cel lines, derived from breast cancer (BrCa), colorectal carcinoma (CRC), head / neck squamous cel carcinoma (HNSCC) and melanoma, as wel as hepatocelular, lung, ovarian, pancreatic and prostate cancer, at concentrations from 0.01 to 10 μM so that IC50 values could be determined (FIG.3P). Among these 40 cel lines, 39 were sensitive to at least one inhibitor with an IC50 of < 10 μM, and 32 lines were sensitive to at least one inhibitor with an IC50 of ≤ 1 μM. Thus, these compounds show broad and potent activity as HIF inhibitors in vitro.

[0391] Compounds SS1.21 and SS3.2 inhibit growth of CRC, HNSCC and BrCa

[0392] Athymic nude mice received a subcutaneous injection of HCT116 CRC cels and when tumors reached a volume of 150 mm3, the mice were administered SS1.21 by intraperitoneal injection (IP) twice daily (BID) for 5 days. Compared to vehicle, SS1.21 significantly inhibited tumor growth (FIG.4A). There was no efect of SS1.21 on mouse appearance, behavior, or body weight (FIG.5A). Immunoblot assays revealed strong expression of HIF-1α and HIF-2α in tumors from vehicle-treated mice but not in tumors from SS1.21-treated mice (FIG.4B). 169 43011.601_P18269-03

[0393] We administered SS1.21 or oxaliplatin, starting when tumors became palpable. Whereas treatment with oxaliplatin, which is a cytotoxic chemotherapy used to treat CRC patients, negatively affected mouse body weight, SS1.21 had no detrimental efect when administered as monotherapy (FIG.5B). SS1.21 was as efective as oxaliplatin in decreasing tumor growth (FIG. 4C) and final tumor weight (FIG.5C), and the combination inhibited tumor growth more than either drug alone (FIG.4C), with no increased toxicity as compared to oxaliplatin alone (FIG.5B).

[0394] To investigate the effect of SS1.21 in immunocompetent mice, CT26 CRC cels were injected subcutaneously into syngeneic Balb / c mice and when tumors reached 100 mm3, the mice were treated with vehicle or SS1.21. SS1.21 inhibited tumor growth (FIG.4D) but had no efect on mouse appearance, behavior or body weight (FIG.5D). Immunoblot assays revealed high HIF- 1α expression in tumors from vehicle-treated mice but not from SS1.21-treated mice (FIG.4E). The luminal area of tumor blood vessels was significantly decreased in SS1.21-treated mice (FIG. 4F and FIG.5E), indicating that the HIFi potently impaired tumor vascularization.

[0395] To investigate the sensitivity of human and mouse HNSCC to SS1.21, we injected FaDu or SCC-VI cels subcutaneously into nude or syngeneic C3H mice, respectively. Treatment with SS1.21 for 5 days inhibited growth of FaDu and SCC-VI tumors (FIG.6A and FIG.6B). FaDu tumors from vehicle-treated mice were dificult to resect due to extensive infiltration of surounding skeletal muscle by cancer cels, whereas tumors from SS1.21-treated mice were easily resected with clean margins (FIG.6C).

[0396] Next, we evaluated the activity of SS3.2 in vivo by performing orthotopic implantation of BT-474 human BrCa cels into the mammary fat pad (MFP) of female nude mice. When tumor volume reached 150 mm3, the mice were treated with SS3.2, which inhibited tumor growth (FIG. 6A) and induced HIF-1α degradation (FIG.6B). The data presented in FIG.3, FIG.4, FIG.5 and FIG.6 indicate that SS1.21 and SS3.2 induce HIF-α degradation and decrease tumor growth in immunodeficient and immunocompetent mouse models of CRC, HNSCC and BrCa.

[0397] Compound 3.2.16 enhances the antitumor activity of anti-CTLA-4 antibody in CRC

[0398] We developed 3.2.16 as a more potent derivative of SS3.2; in addition, 3.2.16 was beter tolerated and could be administered at a higher dose of 40 mg / kg. The nonspecific HIFi echinomycin was reported to potentiate the efect of α-CTLA-4 antibody in an MC38 CRC model (Bailey et al., 2022). We injected MC38 cels subcutaneously into syngeneic C57BL / 6 mice. Once tumors were palpable, mice were treated with vehicle, α-CTLA-4 (200 μg q3D x 5 doses), 3.2.16 170 43011.601_P18269-03(40 mg / kg BID), or both (FIG.4G). Tumors grew rapidly in vehicle-treated mice, which required euthanasia by day 18-23 (FIG.4H and FIG.4I). Tumor growth in mice treated with α-CTLA-4 or 3.2.16 alone was decreased (FIG.4I) but mice required euthanasia by day 36 (FIG.4H). In contrast, 3 out of 5 (3 / 5) mice treated with α-CTLA-4 + 3.2.16 had a complete response (CR) with no palpable tumor after day 21 (FIG.4H and FIG.4I). The mice remained tumor-free despite discontinuation of 3.2.16 on day 38. On day 56, these mice received a second injection of MC38 cels (FIG.4G) and no tumor recurence was observed as of day 130, when the study was terminated (FIG.4H).

[0399] HIFi enhances response to α-CTLA-4: BrCa

[0400] EMT6 triple-negative BrCa (TNBC) cels are resistant to α-CTLA-4 treatment (Khononov et al., 2021; Samanta et al., 2020).1.21S9N was beter tolerated than SS1.21 and, like 3.2.16, could be administered at 40 mg / kg BID. We compared the therapeutic efect of combining α-CTLA-4 with either 1.21S9N or 3.2.16. EMT6 cels were injected into the MFP of syngeneic Balb / c mice and treatment was initiated when tumors became palpable on day 7 (FIG.10A). Compared to control mice, monotherapy increased survival time (FIG.10B) and decreased tumor growth, but the greatest efect was observed with combination therapy, which led to CR in 4 / 10 mice, as compared to 1 / 5 mice treated with α-CTLA-4 alone (FIG.10C-FIG.10H). When these tumor-free animals were rechalenged with EMT6 cels injected into the adjacent MFP on day 70 (FIG.10A), none of the mice developed tumors, in contrast to naïve control mice (FIG.7C). Analysis of RNA from tumors of mice administered HIFi monotherapy revealed decreased expression of markers of angiogenesis (Angptl4, Vegf), T cel exhaustion (Lag3, Tim3), immune checkpoint proteins (B7h3, Pdl1) and other mediators of immunosuppression (Ca9, Cd47, Cd73), whereas Rpl13a expression was unchanged (FIG.10I). HIF-1α levels were markedly decreased in tumors from HIFi-treated as compared to vehicle-treated mice (FIG.10J).

[0401] To further assess compound eficacy, we utilized the MMTV-PyMT geneticaly engineered mouse model, which is widely employed as a preclinical model for TNBC. In this autochthonous model, expression of the polyoma virus middle T antigen (PyMT) driven by the mouse mammary tumor virus (MMTV) promoter leads to the development of palpable tumors starting around day 53 (Atala et al., 2021; Guy et al., 1992). We administered vehicle or 1.21S9N oraly twice daily to MMTV-PyMT female mice beginning on day 53 and monitored tumor growth. Al five vehicle- treated mice developed palpable tumors between days 60 and 75 and required euthanasia by day 171 43011.601_P18269-0394, whereas none of the five mice treated with 1.21S9N developed palpable tumors during the treatment period (FIG.10K and FIG.10L).

[0402] HIFi enhances response to α-CTLA-4: melanoma and prostate cancer

[0403] B16F10 is an aggressive model of immunotherapy-resistant melanoma (Sharma et al., 2019). Pharmacologic or genetic inhibition of HIF activity in B16F10 cels led to improved responses to treatment with a peptide vaccine and α-PD-1 (Lequeux et al., 2021). We treated syngeneic C57BL / 6 mice bearing subcutaneous B16F10 tumors with HIFi, α-CTLA-4, or both (FIG.11A). Although monotherapy delayed tumor growth, combination therapy was synergistic (FIG.11B), with tumor eradication in 3 / 5 mice treated with 1.21S9N and 2 / 5 mice treated with 3.2.16 (FIG.7), and even after a second injection of B16F10 cels on day 57, these mice remained tumor free through day 110, when the study was terminated (FIG.11A and FIG.11C).

[0404] DX1 prostate cancer cels are resistant to combination therapy with androgen receptor inhibitor enzalutamide and α-PD-1 or α-CTLA-4, which was atributed to the expression of another immune checkpoint receptor, B7-H3 (Shi et al., 2023; Zhao et al., 2020). We injected DX1 cels subcutaneously into syngeneic C57BL / 6 male mice, which were treated with α-CTLA-4, HIFi, or both; androgen receptor inhibitor was not administered (FIG.11D). Compared to vehicle, monotherapy had modest efects on tumor growth with CR in 0 / 15 mice (FIG.11E and FIG.11F and FIG.13A-FIG.13F). Treatment with HIFi increased the sensitivity of DX1 tumors to α- CTLA-4, leading to CR in 5 / 10 mice (FIG.11E and FIG.11F and FIG.12E and FIG.12F). Rechalenge with DX1 cels after discontinuation of HIFi therapy (FIG.11D), did not result in tumor growth.

[0405] We also treated 150-mm3 DX1 tumors with vehicle or HIFi for 5 days (FIG.13G) and analyzed mRNA expression 4 hours after the last dose. Analysis of DX1 prostate tumors revealed decreased expression of angiogenic factors (Angptl4, Vegf), immune checkpoint proteins (B7h3, Pdl1), markers of T-cel exhaustion (Lag3, Tim3), and mediators of immune evasion (Ca9, Cd47, Cd73) in tumors from mice treated with HIFi (FIG.13H).

[0406] HIF inhibitors alter the tumor immune cel microenvironment

[0407] We implanted E0771 BrCa cels into the MFP of syngeneic C57BL / 6 female mice. When tumors became palpable on day 7, we started treatment with 3.2.16, α-CTLA-4, or both (FIG.8A, FIG.8B, and FIG.9). Treatment with 3.2.16 or α-CTLA-4 suppressed tumor growth in al mice initialy, but 2 / 5 tumors in 3.2.16-treated mice and 4 / 5 tumors in α-CTLA-4-treated mice 172 43011.601_P18269-03eventualy escaped suppression. In contrast, combination therapy resulted in tumor rejection in 5 / 5 mice (FIG.8E). HIFi treatment was terminated on day 18 and tumors were harvested and weighed. On day 80, mice that remained tumor free were rechalenged with a second injection of E0771 cels into the adjacent MFP (FIG.8C). Naïve animals injected with E0771 cels formed tumors rapidly, whereas none of the mice previously treated with 3.2.16 + α-CTLA-4 developed a tumor after rechalenge. Analysis of E0771 tumors from mice treated with 3.2.16 revealed decreased mRNA expression of markers of angiogenesis (Angptl4, Vegf), T cel exhaustion (Lag3, Pd1, Tim3) and other mediators of immunosuppression (Ca9, Cd47, Cd73, Glut1, IL6, Pdl1), whereas Rpl13a expression was unchanged (FIG.9D).

[0408] The mRNA data from EMT6 and E0771 tumors suggest that treatment of BrCa with HIFi enhances ICB by disrupting multiple mechanisms of immune evasion leading to alterations in the TIME. To test this hypothesis, mice bearing 150-mm3 E0771 tumors were treated with α-CTLA- 4, 3.2.16, or both, and tumors were harvested and analyzed by flow cytometry. The percentages of total CD45+ tumor-infiltrating leukocytes (FIG.9G), CD45+CD3+ T cels (FIG.9H), and CD45+CD3-NK1.1+IFNγ+ cytolytic NK cels (FIG.9I) were al increased by treatment with 3.2.16, either alone or in combination with α-CTLA-4. In contrast, the percentages of CD45+CD11b+F4 / 80+ tumor-associated macrophages (TAMs) (FIG. 9J) and CD45+CD11b+Ly6G+ myeloid-derived suppressor cels (MDSCs) (FIG.9K) were decreased in tumors from mice treated with 3.2.16 or 3.2.16 + α-CTLA-4. The percentage of CD45-CD3- PDL1+ cancer cels was decreased in al three treatment groups (FIG.9L). These data indicate that 3.2.16 + α-CTLA-4 converted an immunosuppressive TIME, dominated by MDSCs and TAMs, into one favoring anti-tumor immunity, populated by NK and T cels.

[0409] Efect of HIF inhibitors on response to immunotherapy: a meta-analysis

[0410] Our experimental strategy was to survey the efect of HIFi therapy on the response to ICB in syngeneic mouse models of melanoma, BrCa, CRC, and prostate cancer. Because the number of mice in each individual treatment group was smal, we performed a meta-analysis combining the results of al cancer types and treatment protocols. The aggregate CR to ICB monotherapy was 1 / 30 (3.3%), as compared to 24 / 45 (53%) in mice treated with ICB + HIFi (Table 1 and FIG.16). 173 43011.601_P18269-03Table 1. Aggregate complete response (CR) rates. Mice were treated with ICB alone (α-CTLA-4 [C] or α-PD-1 [P]) or in combination (Comb) with

[0411] Eficacy and safety of dual HIF-1 / 2 vs selective HIF-2 inhibition

[0412] We found that tumor-bearing mice tolerated oral administration of HIFi at doses as high as 180 mg / kg twice daily without adverse efects on appearance, behavior, or body weight.

[0413] Treatment of CT26 colorectal cancer cels with the HIF-2 inhibitor PT2385 inhibited hypoxia-induced expression of Adm but not Ca9 mRNA, whereas 1.21S9N inhibited both. PT2385 (60 mg / kg, once daily) was active in a mouse model of RCC (Walace et al., 2016). We administered PT2385 at this dose to CT26 tumor-bearing mice.1.21S9N was administered at the same dose either once or twice daily. PT2385 treatment resulted in 30% tumor growth inhibition, whereas 1.21S9N treatment led to 70% tumor growth inhibition as monotherapy (FIG.17A). We tested whether 1.21S9N or PT2385 potentiated the efect of anti-PD-1 antibody. Treatment with 1.21S9N also increased survival when combined with α-PD-1 therapy, with a CR rate of 40% for 1.21S9N + α-PD1 compared to 0% for PT2385 + α-PD1 (FIG.17B).

[0414] Analysis of tumor tissue demonstrated that 1.21S9N monotherapy inhibited expression of mRNAs encoding the angiogenic factors Adm, Angptl4, Pgf and Vegf, as wel as the immune checkpoint receptors Pd-l1 and Cd47, whereas PT2385 only inhibited Adm and Vegf (FIG.17C). Tumors harvested from 1.21S9N-treated mice showed decreased vascularization compared to 174 43011.601_P18269-03tumors treated with vehicle or PT2385 (FIG.17D). Thus, dual HIF-1 / 2 inhibition has greater efects on gene expression, leading to enhanced suppression of tumor growth and vascularization.

[0415] Prior studies in mice revealed that PT2385 inhibits ventilatory responses to hypoxia (Cheng et al., 2020). We treated mice with PT2385 or 1.21S9N at the same dose and schedule (30 mg / kg q12h x 3 doses). Whole body plethysmography revealed disordered breathing with increased apnea episodes (cessation of breathing) in mice treated with PT2385 as compared to vehicle, whereas 1.21S9N had no adverse efect on breathing (FIG.17E and FIG.17F). Thus, head-to-head testing in mice indicates superior eficacy (FIG.8A-FIG.8D) and safety (FIG.17E and FIG.17F) of the dual HIF-1 / 2 inhibitor 1.21S9N compared to the HIF-2-selective inhibitor PT2385.

[0416] Compound 3.2.16 enhances the antitumor activity of α-CTLA-4 in breast cancer

[0417] Compound 3.2.16 inhibited HIF target gene expression with an IC50 of 0.13 mM in E0771 cels (FIG.3P). We implanted E0771 cels into the MFP of syngeneic C57BL / 6 female mice. When tumors became palpable on day 7, we started treatment with 3.2.16 (40 mg / kg BID, days 7-18), α- CTLA-4 (200 mg, q3D, days 7, 10, 13, 16), or both (FIG.9A). Compared to vehicle-treated mice, treatment with 3.2.16 or α-CTLA-4 suppressed tumor growth in al mice initialy, but 3 of 5 tumors in 3.2.16-treated mice and 4 of 5 in the α-CTLA-4-treated mice eventualy escaped suppression (FIG.9B, FIG.9C, FIG.9E, and FIG.9F). In contrast, the combination of 3.2.16 and α-CTLA-4 resulted in tumor rejection of 5 out of 5 mice (FIG.9E). HIF inhibitor (HIFi) treatment was terminated on day 18 and on day 80, two of the animals remained tumor free and were rechalenged with a second injection of E0771 cels into the adjacent MFP (FIG.9A). As a control, an additional group of naïve animals were injected with E0771 cels and tumors formed rapidly in these mice (FIG.9D). As observed in the MC38 CRC model, neither of the mice previously treated with 3.2.16 + α-CTLA-4 developed a tumor after rechalenge. RT-qPCR analysis of E07761 tumors from mice treated with 3.2.16 revealed significantly decreased expression of markers of angiogenesis (Angptl4, Vegfa), T cel exhaustion (Lag3, Pd1, Tim3), immune checkpoint proteins (B7h3, Pdl1) and other mediators of immunosuppression (Ca9, Cd47, Cd73, Glut1, IL6, Vegfa), whereas expression of Rpl13a (control, non-HIF-regulated) RNA was unchanged (FIG.9Dd).

[0418] A mouse triple-negative breast cancer, EMT6, which is resistant to α-CTLA-4 treatment, Jure-Kunkel et al., 2013. was also investigated. We compared the therapeutic efect of combining α-CTLA-4 with either 1.21S9N or 3.2.16, which inhibited HIF target gene expression in these cels with IC50 values of 0.3 and 0.5 mM, respectively (FIG.3P).1.21S9N was beter tolerated than 175 43011.601_P18269-03SS1.21 and, like 3.2.16, could be administered at 40 mg / kg BID. EMT6 cels were injected into the MFP of syngeneic Balb / c mice and treatment was initiated when tumors became palpable on day 7 (FIG.10A). Compared to control mice, monotherapy (1.21.S9N, 3.2.16, or α-CTLA-4) increased survival time and decreased tumor growth but the greatest efect was observed with combination therapy, which led to CR in 2 of 5 mice treated with α-CTLA-4 and HIFi, as compared to 1 of 5 mice treated with α-CTLA-4 alone (FIG.10B-FIG.10H). When these tumor-free animals were rechalenged with implantation of EMT6 cels into the adjacent MFP on day 70, 25 days after discontinuation of HIFi therapy (FIG.10A), none of the mice developed tumors in contrast to naïve control mice (FIG.7C).

[0419] Analysis of RNA from EMT6 tumors of mice treated with HIFi revealed significantly decreased expression of markers of angiogenesis (Angptl4, Vegfa), T cel exhaustion (Lag3, Tim3), immune checkpoint proteins (B7h3, Pdl1) and other mediators of immunosuppression (Ca9, Cd47, Cd73, Vegfa), whereas Rpl13a expression was unchanged (FIG.10I). HIF-1α levels were markedly decreased in tumors from mice treated with HIFi (FIG.10J). The RNA data from E0771 and EMT6 tumors suggest that treatment of breast cancer with HIFi disrupts multiple mechanisms of immune evasion to enhance the anti-tumor activity of ICB.

[0420] Treatment of mice bearing 150-mm EMT6 orthotopic breast tumors for 5 days with 1.21S9N (40 mg / kg BID), 3.2.16 (40 mg / kg BID), or 3.2n (20 mg / kg BID) monotherapy significantly inhibited tumor growth. Treatment with 3.2.67n (40 mg / kg BID) monotherapy decreased EMT6 tumor growth but the efect was not statisticaly significant (FIG.24A). Tumors from mice treated with any one of these four HIF inhibitors showed a marked loss of HIF-1a protein expression (FIG.24B). Treatment of mice bearing HCC1954 human HER2+ orthotopic breast tumors with 3.2n or 3.2.16n monotherapy for 7 days significantly inhibited tumor growth (FIG.24C).

[0421] Compounds 1.21.S9N and 3.2.16 enhance antitumor activity of α-CTLA-4 in melanoma

[0422] B16F10 is an aggressive mouse model of immunotherapy-resistant melanoma. van Elsas et al., 1999. A previous study reported that pharmacologic or genetic inhibition of HIF activity in B16F10 cels led to improved responses to treatment with a peptide vaccine and α-PD-1. Lequeux et al., 2021. Treatment with 1.21S9N or 3.2.16 inhibited HIF target gene expression in hypoxic B16F10 cels with IC50 values of 0.08 and 0.10 mM, respectively (FIG.3P). We treated syngeneic C57BL / 6 mice bearing subcutaneous B16F10 tumors with 1.21S9N, 3.2.16 or α-CTLA-4, either 176 43011.601_P18269-03alone or in combination (FIG.11A). Although monotherapy delayed tumor growth, combination therapy was synergistic, with tumor eradication in 3 of 5 mice treated with 1.21.S9N + α-CTLA- 4 and 2 of 5 mice treated with 3.2.16 + α-CTLA-4, and even after a second injection of B16F10 cels on day 57, these mice remained tumor free through day 110, when the study was terminated (FIG.11A-FIG.11C and FIG.12).

[0423] Compound 1.21.S9N enhances antitumor activity of α-PD-1 against Lewis lung carcinoma

[0424] Lewis lung carcinoma (LLC), which is resistant to anti-PD-1 therapy, Ueha et al., 2015, was sensitive to 1.21.S9N in vitro with IC50 = 0.2 mM (FIG.3P). Syngeneic C57BL / 6 mice with subcutaneous LLC tumors were treated with 1.21.S9N (days 6-51), α-PD-1 (days 6, 9, 12, 15, 18), or both (FIG.25A). LLC was not responsive to either monotherapy; in contrast, CR or stable disease (SD) was achieved in 5 of 5 mice treated with combination therapy (FIG.25B-FIG.25F). Mice with a CR received a second injection of LLC cels on day 62 and remained tumor-free through day 120 when the study was terminated. Thus, HIF inhibitors improve the response to α- PD-1, as wel as α-CTLA-4 therapy.

[0425] Compound 3.2.16 enhances α-CTLA-4 or α-PD-1 antitumor activity in pancreatic cancer

[0426] Pancreatic cancer is known for high mortality rates, with a 5-year overal survival of 10%, and near-universal resistance to ICB. Hu and O’Reily, 2024. The HIF-2-selective inhibitor PT- 2399, however, has been shown to inhibit pancreatic tumor growth when administered in combination with α-CTLA-4. Garcia Garcia et al., 2022.

[0427] LSL-KrasG12D; LSL-Trp53R172H; PdxCre conditional knockout mice develop pancreatic ductal adenocarcinoma, which led to establishment of the KPC tumor cel line. Hingorani et al., 2005. KPC cels were sensitive to 3.2.16, with an IC50 = 0.1 mM (FIG.3P). Syngeneic C57BL / 6 mice were injected subcutaneously with KPC cels and then treated with 3.2.16, immune checkpoint blockade (ICB; α-PD-1, or α-CTLA-4), or 3.2.16 with either α-PD-1 or α-CTLA-4 (FIG.26A). Compared to monotherapy, combination therapy with 3.2.16 and ICB significantly inhibited tumor growth: 5 of 10 mice treated with HIFi + ICB had SD compared to 0 of 15 mice treated with monotherapy (FIG.26A-FIG.26G).

[0428] Immunohistochemical staining of tumor sections with an antibody against the vascular endothelial cel marker CD31 revealed decreased tumor vessel area in mice treated with 3.2.16 compared to vehicle control (FIG.26H). Tumors from vehicle-treated mice were highly invasive 177 43011.601_P18269-03and could only be excised with surounding muscle tissue, whereas tumors from 3.2.16-treated mice were easily separated from the surrounding tissue.

[0429] Compounds 1.21.S9N and 3.2.16 enhance α-CTLA-4 antitumor activity in prostate cancer

[0430] The mouse prostate cancer cel line DX1 is resistant to combination therapy with androgen receptor inhibitor enzalutamide and α-PD-1 or α-CTLA-4, which was atributed to the expression of another immune checkpoint receptor, B7-H3. Shi et al., 2023. Treatment of DX1 cels with 1.21S9N or 3.2.16 inhibited hypoxia-induced HIF target gene expression with IC50 values of 0.2 and 0.3 mM, respectively (FIG.3P).

[0431] We found that B7-H3 mRNA expression was hypoxia-induced in DX1 cels and inhibited in a dose-dependent manner by treatment with 1.21S9N or 3.2.16 (FIG.27A). We injected DX1 cels subcutaneously into syngeneic C57BL / 6 male mice, which were treated with α-CTLA-4 (days 7, 10, 13, 16, 19, 22), or 1.21S9N or 3.2.16 (days 7-48), either alone or in combination; androgen receptor inhibitor was not administered (FIG.26I). Compared to vehicle, monotherapy had only modest effects on tumor growth with CR in 0 of 15 mice (FIG.13A-FIG.13D). Treatment with HIFi, however, increased the sensitivity of DX1 tumors to α-CTLA-4, leading to complete tumor regression in 5 of 10 mice (FIG.13E-FIG.13F). Rechalenge with DX1 cels on day 66, which was 18 days after discontinuation of HIFi therapy (FIG.26I), did not result in tumor growth.

[0432] We also treated 150-mm3 DX1 tumors with vehicle, 1.21S9N, or 3.2.16 for 5 days (FIG. 27B) and analyzed mRNA expression 4 hours after the last dose. As observed for E0771 (FIG. 9D) and EMT6 (FIG.10I) breast tumors, analysis of DX1 prostate tumors revealed significantly decreased expression of angiogenic factors (Angptl4, Vegfa), immune checkpoint proteins (B7h3, Pdl1), markers of T-cel exhaustion (Lag3, Tim3), and mediators of immune evasion (Ca9, Cd47, Cd73, Vegfa) in tumors from mice treated with HIFi (FIG.13H). Expression of mRNA encoding the immune checkpoint protein B7h3 was induced when DX1 cels were cultured at 1% O2 and this induction was inhibited by treatment with 1.21S9N or 3.2.16 (FIG.27A).

[0433] Treatment of mice bearing PC3 human prostate cancer xenografts with 1.21S9N (40 mg / kg BID) or 3.2.16 (40 mg / kg BID) for 5 days significantly inhibited tumor growth. Treatment with 3.2n (20 mg / kg BID) decreased tumor growth but the effect was not statisticaly significant (FIG. 27B).

[0434] Inhibition of Choroidal Neovascularization by Compound 3.2.16. We previously reported that the dual HIF-1 / HIF-2 inhibitor 32-134D was effective at blocking neovascularization 178 43011.601_P18269-03folowing laser-induced injury to the mouse cornea (Zhang et al., 2023). Because it is not clear whether 32-134D has sufficient drug-like properties to enable its use in the clinic, we have tested 3.2.16 head-to-head with 32-134D. As previously described, C57BL / 6 mice received laser injury to the choroid on day 0 and on day 3 received a 1-mL intravitreal injection of vehicle (DMSO), 32-134D (70 ng), or 3.2.16 (70 or 140 ng). On day 7, the mice were euthanized, enucleated, and the eyes stained with fluorescently-labeled isolectin B4, which binds selectively to neovasculature. For each of the four treatment conditions, four eyes were analyzed, with 3 or 4 laser injury sites per eye. The area of choroidal neovascularization (CNV) was calculated for each injury site and the data were analyzed for diferences between groups using the 2-tailed Student’s t test. The CNV lesion area (presented in FIG.29) as mean + standard deviation) was significantly decreased by treatment with 32-134D or 3.2.16 compared to vehicle control (VE) at 70 ng (P < 0.01) and by treatment with 3.2.16 at 140 ng (P < 0.001).3.2.16 was as efective as 32-134D at the same dose (70 ng) and was as or more effective at the higher dose (140 ng). From these results, we conclude that 3.2.16 is an efective agent for inhibiting ocular neovascularization.

[0435] Discussion

[0436] There are over 100 publications in which immunohistochemical analysis of tumor biopsies has established an association between HIF-1α or HIF-2α expression and patient mortality (Wicks and Semenza, 2022), including lung, colorectal, pancreatic, breast, and prostate cancer, which are the major causes of cancer mortality in the U.S., accounting for greater than 300,000 deaths annualy (Siegel et al., 2024). The association between intratumoral HIF-1 / 2α expression and cancer mortality is indicative of a large body of data demonstrating key roles for HIFs in cancer progression (Cowman and Koh, 2022; Rankin et al., 2016; Sitkovsky et al., 2014; Wicks and Semenza, 2022; Yuan et al., 2024). The limited therapeutic eficacy of ICB reflects multiple mechanisms by which cancer cels evade detection and kiling by immune cels, and many of these are mediated by HIF target gene products (Chouaib et al., 2012; Semenza, 2021; Sitkovsky et al., 2014).

[0437] HIFi therapy induced profound changes in the intratumoral expression of key regulators of innate and adaptive immunity, and reprogrammed the TIME from one dominated by immunosuppressive MDSCs and TAMs to one dominated by NK and T cels capable of mediating anti-tumor immunity. The decreased expression of mRNAs encoding the immune checkpoint proteins B7h3, Lag3, Pdl1, and Tim3 in response to HIFi therapy was striking and represents the 179 43011.601_P18269-03equivalent of treating with multiple ICB antibodies. Thus, HIFi therapy increased T / NK cel numbers and decreased T / NK cel exhaustion. The increased CR to ICB associated with HIFi therapy across a broad sampling of cancer types suggests that this combination wil have widespread clinical utility. In mice that responded to combination therapy, the immune system continued to monitor for cancer recurence after termination of therapy and responded to rechalenge such that the animals remained cancer free.

[0438] We found that treatment of CRC tumor-bearing mice with HIFi had a profound anti- angiogenic effect, with a markedly decreased luminal diameter of tumor blood vessels that resulted from the decreased expression of mRNAs encoding ANGPTL4, VEGF and other angiogenic factors. Treatment of HNSCC tumor-bearing mice with HIFi led to major changes in the invasive properties of the tumors such that they were easily and cleanly excised, whereas tumors from vehicle-treated mice showed extensive infiltration of the surounding tissue making clean excision dificult. Thus, dual HIF-1 / 2 inhibition has profound efects on tumor biology.

[0439] The carotid body (CB) senses hypoxemia and stimulates ventilation, a fundamental homeostatic response to hypoxia, which is inhibited by PT2385 (Cheng et al., 2020). Mice that are heterozygous for a HIF-2α nul alele have significantly increased apnea episodes (Peng et al., 2011). In mice exposed to intermitent hypoxia, HIF-2α levels in the CB decreased (Nanduri et al., 2009) whereas HIF-1α levels increased (Peng et al., 2006), suggesting that these factors may have opposing efects in the CB. In clinical trials, an adverse efect of PT2399 / belzutifan has been hypoxemia (Falah et al., 2024). Selective HIF-2α inhibition may have adverse effects on ventilation that are not observed when both HIF-1α and HIF-2α are targeted.

[0440] The compounds we identified were surprising in several regards. First, SS1.21 and 1.21S9N bind to the bHLH domain of HIF-1α and HIF-2α. We are not aware of any other smal molecule that targets this domain in any transcription factor. Second, SS3.2 and 3.2.16 bind to the PAS-B subdomain of HIF-1α and HIF-2α. While other compounds (PT2385 and PT2399) bind to the PAS-B subdomain of HIF-2α, those compounds do not bind to HIF-1α (Walace et al., 2016). Thus, the interaction of SS3.2 or 3.2.16 with the PAS-B subdomain difers from that of PT2385 or PT2399. The existence of multiple HIF inhibitors with distinct molecular binding sites or binding modes wil be advantageous in the therapeutic response to acquired drug resistance arising from mutations that alter the drug binding site, as exemplified by the multiple generations of BCR- ABL inhibitors that are used to treat leukemia (Alves et al., 2021). 180 43011.601_P18269-03

[0441] Materials and Methods

[0442] Study Design and Approvals

[0443] Sample size was n = 3 for analysis of RNA expression in cultured cels and n = 5 mice for tumor studies, except for flow cytometry studies where n = 5-10, with lesser number due to technical failure with one antibody (anti-CD3); these were the only data excluded in the study. Al outliers were included. Endpoint: tumor-bearing mice were euthanized after 5 days of treatment or when tumor diameter exceeded 1.5 cm; the tumor became ulcerated; or the animal showed any sign of distress. The objective of the study was to identify smal molecule HIF inhibitors and demonstrate their efect on tumor growth alone and in combination with ICB. Al mice were randomized prior to treatment and tumor dimensions were measured in a blinded manner. Procedures involving mice were conducted in accordance with the guidelines outlined in the NIH Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). Protocols were reviewed and approved by the Johns Hopkins University Animal Care and Use Commitee.

[0444] Site Identification by Ligand Competitive Saturation (SILCS) Screening of HIF-2α

[0445] SILCS simulations (Guvench and MacKerel, 2009; Raman et al., 2011) were performed using the X-ray crystal structure of the HIF-2α:HIF-1β complex (PDB 4ZP4; Wu et al., 2015). The HIF-2α protein structure, modeled to generate coordinates of missing residues in the crystal structure (residues 11-25, 150-161, 202-218) using Modeler (Fiser and Sali, 2003), was immersed in an aqueous solution of solutes at approximately 0.25 M representative of diferent functional groups (benzene, propane, formamide, methanol, dimethylether, imidazole, acetate, and methylammonium representative of aromatic, aliphatic, hydrogen bond donor, hydrogen bond acceptors, negatively and positively charged groups). The solutes and water were subjected to oscilating excess chemical potential (μex) Grand Canonical Monte Carlo / Molecular Dynamics (GCMC / MD) simulations (Lakkaraju et al., 2014) to sample the distribution of solutes and water in and around the protein.

[0446] From these simulations, probability distributions of solutes and water were determined by counting their occurence on grid points defined as 1 Å3 voxels that encompass the protein structure, normalized to the concentration of solutes in aqueous solution, and Boltzmann transformed (Lakkaraju et al., 2015) to yield 3D distributions of GFEs of functional groups around the protein, termed GFE FragMaps. GCMC / MD simulations were performed using in-house GCMC code (Lakkaraju et al., 2015) with MD performed using the Gromacs package (Abraham 181 43011.601_P18269-03et al., 2015). Force field parameters were the CHARMM36m protein force field (Huang et al., 2017) with the CHARMM TIP3P water model (Jorgensen, 1981) and the CHARMM General Force Field (CGenFF; Vanommeslaeghe et al., 2010) used for the solutes, fragments and ligands (Ustach et al., 2019; Goel et al., 2021).

[0447] Identification of putative binding sites on HIF-2α utilized the SILCS-Hotspots approach (MacKerel et al., 2020). A set of smal mono- and bi-cyclic ring fragments that commonly occur in known drugs (Taylor et al., 2014) were docked using SILCS-MC approach into the FragMaps encompassing the entire protein structure. Spatial clustering was performed for individual fragments folowed by clustering over al fragment types to identify binding sites encompassing one or more fragments, caled Hotspots. Three putative binding sites were chosen based on the presence of 2 or more adjacent Hotspots, along with FragMaps, protein surface and exclusion map data.

[0448] Pharmacophore features were generated at each site using the SILCS-Pharm protocol (Yu et al., 2015). Virtual database screening was performed by selecting multiple pharmacophore hypotheses for each site that contained a subset of 3 or 4 of the total number of features in each site in various spatial 3D relationships. The 3 and 4-point pharmacophore hypotheses were al individualy screened against an in-house database that contains 54,359 compounds from Maybridge; 710,933 compounds from Chembridge and 3,114 FDA approved drugs. For each individual pharmacophore hypothesis al compounds with the corect number and type of functional groups that were spatialy within a root-mean-square difference ≤ 1.2 Å of the features were considered hits with a maximum of 10,000 being selected. Al hits from the individual pharmacophores for each site were then pooled for further analysis.

[0449] The hit compounds were subjected to free energy scoring based on the SILCS-MC approach to generate ligand GFE (LGFE) scores. Calculating LGFE involves classification of the ligand atoms into FragMap type, folowed by the summation of GFEs at the coordinate position of the classified ligand atoms (Ustach et al., 2019; Goel et al., 2021). SILCS-MC involves sampling the orientation and conformation of the ligand in the field of the FragMaps, based on LGFE score along with the intramolecular energy of the ligand based on the CGenFF function with a 1 / r dielectric constant (Vanommeslaeghe et al., 2010). SILCS-MC pose refinement was performed starting from the docked orientation of each ligand from the pharmacophore search. MC sampling involved 100 steps of translations, molecular rotations, and dihedral rotations of 0.5 182 43011.601_P18269-03Å, 15^ and 180^, respectively folowed by MC simulated annealing of 1000 steps of 0.25 Å, 9^ and 9^, respectively, for a minimum of 50 runs based on a convergence of 0.5 kcal / mol up to a maximum of 250 runs. Five repeats of this protocol were performed at each site and binding pose and orientation of ligands were ranked based on most negative LGFE scores.

[0450] Final compound selection considered physical properties indicative of drug-likeness. LogP(o / w), number of H-bond donors and acceptors, and molecular mass were obtained for the selected compounds using MOE (Chemical Computing Group).4-dimensional bioavailability (4DBA) values (Oashi et al., 2015) were calculated which combines Lipinski’s rule of five (Lipinski, 2000) into a single scalar value. Ligands with molecular weight < 200 and > 550 Daltons were discarded. Ligands that did not pass the PAINS rules (Bael and Holoway, 2010) indicating potential toxicity were also removed. The remaining ligands were grouped into clusters based on chemical similarity using Morgan fingerprinting (cutof = 0.45) with compounds for biological evaluation obtained by choosing ligands with the most negative LGFE scores in each cluster. Virtual database screening was performed targeting each site involving first the pharmacophore screening folowed by final ranking based on the SILCS-MC LGFE scores. For each site, the top 1000 ranked ligands from each database were selected for final filtering based on physical properties, including Lipinski’s rule of 5 and the 4DBA metric. From this procedure 100 compounds were selected for each site.

[0451] Cel culture

[0452] The source of cel lines and culture media are shown in Table 4. Cel line authentication and Mycoplasma testing were performed at the Johns Hopkins University Genetic Resources Core Facility. Other cel culture reagents are shown in Table 5.

[0453] Cel-based screening assay

[0454] The compounds for cel-based screening were purchased from Molport and dissolved in DMSO. Hep3B-c1 cels were plated onto 24-wel plates and, the folowing day, cels were exposed to either 20% or 1% O2 for 24 hours in the presence of vehicle (DMSO) or chemical compound (Salman et al., 2022). FLuc and RLuc activity in cel lysates was determined using the Dual- Luciferase Reporter Assay System (Table 5) and a VICTOR Nivo plate reader. Hep3B cels were transiently transfected with pSV-RL, HIF-2α expression vector, and pEPO-ProEn-FL (Dioum et al., 2009), and 24 hours later, were exposed to hypoxia.

[0455] RT-qPCR assays 183 43011.601_P18269-03

[0456] Cels were cultured in 6-wel plates overnight (see Table 4 for seeding density), pre-treated for 1 hour with compound, exposed to 20% or 1% O2 for 24 hours, and lysed in TRIzol reagent (Table 5). Tumor tissue was flash frozen in liquid nitrogen and RNA was extracted using TRIzol. RNA was reverse transcribed to cDNA using the High Capacity RNA-to-cDNA Kit, and qPCR was performed using SYBR Green qPCR Master Mix and the CFX96 Real-Time PCR Detection System (BioRad) with primers listed in Table 6. The expression of target mRNA (E) was normalized to 18S rRNA using the threshold cycle (Ct) method and expressed as fold change: E = 2–Δ(ΔCt), where ΔCt = Cttarget gene – Ct18S rRNA and Δ(ΔCt) = ΔCttreatment – ΔCtcontrol. To determine IC50 values, we analyzed expression of several HIF target genes in each cel line and chose the gene with highest fold induction when that cel line was exposed to 1% O2 for 24 hours in the absence of HIFi.

[0457] Immunoprecipitation assays

[0458] Soluble lysates were prepared from cultured cels in lysis bufer (50 mM HEPES [pH 7.9], 150 mM NaCl, 1 mM EDTA, 10% glycerol, 1% IGEPAL, 1 mM PMSF supplemented with complete protease inhibitor cocktail) and debris was peleted by centrifugation at 13,000 rpm for 20 minutes. The supernatant was precleared by incubation with protein G-Sepharose (Table 5) for 1 hour, an aliquot was reserved for input analysis, and remaining lysate was incubated with antibody overnight at 4°C. Protein G-Sepharose was added to the samples and incubated for 4 hours at 4°C. Immunoprecipitates were washed 3 times with lysis bufer and subjected to immunoblot assay using antibodies listed in Table 5.

[0459] Immunoblot assays

[0460] Cultured cels or tumor tissue was lysed in RIPA bufer. Proteins were fractionated by 8- 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocelulose membranes, which were blocked with 5% (w / v) nonfat milk / 0.1% Tween 20 in PBS for 1 hour and incubated at 4°C overnight with primary antibody (Table 5) in blocking solution. Membranes were incubated with HRP-conjugated secondary antibody for 1 hour. Chemiluminescent signal was generated using ECL Prime reagent and detected using the ChemiDoc Imaging System (BioRad).

[0461] Tumor studies

[0462] Cancer cels were trypsinized, rinsed with PBS and injected into mice in 100 ml of a 1:1 Matrigel:PBS suspension (Table 5). The number of cels injected, site of injection and strain / sex of recipient mice are shown in Table 7. Tumor long (L) and short (S) axis were measured (in mm) 184 43011.601_P18269-03using electronic calipers and tumor volume (V) was calculated: V (mm3) = (L × S2) / 2. Drugs were formulated for intraperitoneal administration as described in Table 8. Mice were euthanized when tumor diameter reached 1.5 cm, tumors became ulcerated, or mice showed signs of distress. For rechalenge studies, cels were implanted in the adjacent MFP or contralateral flank relative to the initial injection.

[0463] Measurement of Breathing

[0464] Breathing was monitored by whole body plethysmograph (SCIREQ, Montreal, QC, Canada) in unsedated 2-month-old male C57 / BL6 male mice (Jackson Laboratory) breathing room air as described previously (Nanduri et al., 2009; Peng et al., 2011). Al measurements were made between 1:30 PM and 3:30 PM to exclude any confounding influence of circadian variations. Al measurements were made at an ambient temperature of 25°C ± 1°C. One hour was alowed for acclimation of mice to the plethysmography chamber, and breathing was then monitored for 2 hours. Apnea is defined as cessation of breathing longer than the duration of 3 normal breaths. Apneas were scored with a commercial program (IOX, SCIREQ). Snifs, post-sigh apneas, and movement artifacts were excluded from analysis.

[0465] Flow Cytometry

[0466] Tumors were minced and digested using colagenase (1 mg / mL) at 37°C for 30 minutes. The resulting single cel suspension was passed through a 70-μm strainer and rinsed twice with cold PBS. Cels were resuspended in Fc Block (BD Biosciences) and stained with multiple fluorescent antibodies (Table 9) to characterize specific immune cel populations (Salman et al., 2022; Samanta et al., 2020). Cels were processed for flow cytometry analysis using FACS Diva software (BD). Live cels were identified and gated based on side-scater and forward-scater plots. Gating strategies were employed using unstained control, fluorescence minus one and single- stained cel samples (FIG.13D). FlowJo software was used for data analysis.

[0467] Tumor immunohistochemistry

[0468] Tumor tissues were fixed in 10% formalin and paraffin embedded for sectioning. Tissue sections were deparafinized, hydrated, and immersed in antigen retrieval solution (Table 5). Sections were blocked with 10% normal goat serum for 1 hour; incubated with anti-CD31 antibody overnight at 4°C; treated with 3% H2O2 for 10 minutes; incubated with SignalStain Boost Detection Reagent for 30 minutes at room temperature; washed; and covered with SignalStain DAB folowed by hematoxylin counterstaining. Sections were dehydrated and mounted with 185 43011.601_P18269-03coverslips. For analysis, three fields at 20x magnification from three diferent tumors were captured using ImageJ (NIH).

[0469] Statistical Analysis

[0470] Al analyses were performed using Prism 9 (GraphPad Software). Significant diferences between groups were determined using Student’s t-test or analysis of variance (ANOVA) with multiple comparisons. Kaplan–Meier survival plots were analyzed by log-rank test. EXAMPLE 2 Supplementary Materials

[0471] Site Identification by Ligand Competitive Saturation (SILCS) Screening of HIF-2α

[0472] SILCS simulations, Guvench and MacKerel, 2009; Raman et al., 2011, were performed using the X-ray crystal structure of the HIF- 2α:HIF-1β complex (PDB 4ZP4; Wu et al., 2015) to evaluate the functional group free energy afinity patern in and around the protein using a range of chemicaly diverse solutes. In the SILCS simulations, the HIF-2α protein structure, modeled to generate coordinates of missing residues (residues 11-25, 150-161, 202-218) in the crystal structure using Modeler, Fiser and Sali, 2003, was immersed in an aqueous solution of solutes at approximately 0.25 M representative of diferent functional groups (benzene, propane, formamide, methanol, dimethylether, imidazole, acetate, and methylammonium representative of aromatic, aliphatic, hydrogen bond donor, hydrogen bond acceptors, negatively and positively charged groups). The solutes and water were subjected to oscilating excess chemical potential (μex) Grand Canonical Monte Carlo / Molecular Dynamics (GCMC / MD) simulations, Lakkaraju et al., 2014, to sample the distribution of solutes and water in and around the protein. From these simulations, probability distributions of the solutes and water were determined by counting their occurence on grid points defined as 1 Å3 voxels that encompass the protein structure. These probabilities were normalized with respect to the concentration of the solutes in aqueous solution and Boltzmann transformed, as previously described, Lakkaraju et al., 2015, to yield 3D distributions of grid free energies (GFEs) of the diferent functional groups around the entire protein, termed GFE FragMaps. Regions that are favorable for functional group binding relative to being in solution acquire negative GFE scores while unfavorable regions acquire positive GFE values. GFE values of zero are equivalent to the group being in aqueous solution. In addition, an exclusion map is generated defined as regions of the protein not sampled by solutes or water during the SILCS 186 43011.601_P18269-03simulations, representing a strictly inaccessible surface. FIG.14a and FIG.14b show HIF-2α with SILCS FragMaps and exclusion map.

[0473] GCMC / MD simulations were performed using in-house GCMC code, Lakkaraju et al., 2014, with MD performed using the Gromacs package. Abraham et al., 2015. Force field parameters were the CHARMM36m protein force field, Huang et al., 2017, with the CHARMM TIP3P water model, Jorgensen, 1981, and the CHARMM General Force Field (CGenFF; Vanommeslaeghe et al., 2010), used for the solutes, fragments and ligands. Details of the SILCS simulations, including truncation schemes, may be accessed as published (Ustach et al., 2019; Goel et al., 2021). Identification of putative binding sites on HIF-2α applied the SILCS-Hotspots approach. MacKerel et al., 2020. It this approach a set of smal mono- and bicyclic ring fragments that commonly occur in known drugs, Taylor et al., 2014, are comprehensively docked using the SILCS-MC approach into the FragMaps encompassing the entire protein. Spatial cluster is then performed for the individual fragments folowed by clustering over al fragment types to identify binding sites encompassing one or more fragments, caled Hotspots. Based on visual inspection of the Hotspots focusing on those with 2 or more adjacent Hotspots, along with the FragMaps, protein surface and exclusion maps, three putative binding sites were chosen in the bHLH domain (site 1), the PAS-A subdomain (site 2), and the PAS-B subdomain (site 3) of HIF-2α. For virtual screening of large databases of ligands, pharmacophore features were generated in each site using the SILCS- Pharm protocol. Yu et al., 2015. Pharmacophore features are models that identify the types of functional groups and the spatial relationship between those features that drug-like molecules should have to bind to a specific site. FIG.14c shows pharmacophore features at each of the three sites on HIF-2α. Virtual database screening was then performed by selecting multiple pharmacophore hypotheses for each site that each contained a subset of 3 or 4 of the total number of features in each site in various spatial 3D relationships. For example, site 3 contains seven apolar, three H-bond acceptor, and one positively-charged feature (Table 2). Table 2. Pharmacophore features obtained for each site using SILCS-Pharm. m187 43011.601_P18269-03Table 2. Pharmacophore features obtained for each site using SILCS-Pharm. Site 1 Site 2 Site 3m

[0474] Eight diferent sets of pharmacophore features were generated for site 3 (Table 3), each containing diferent combinations of apolar and polar features with each site containing a minimum of 2 apolar features. The advantage of the use of multiple pharmacophores for each site is that it alows for ligands with diferent colections and spatial dispositions of functional groups to be selected. This approach was also applied to the other sites, with six pharmacophores being selected each for site 1 and site 2 (FIG.14c). The 3 and 4-point pharmacophore hypotheses were 188 43011.601_P18269-03al individualy screened against an in-house database that contains 54,359 compounds (114,506 protomers) from Maybridge; 710,933 compounds (1,664,178 protomers) from Chembridge and 3,114 compounds (6,228 protomers) from FDA approved drugs. The screening of FDA, Chembridge and Maybridge databases was performed separately for each site. For each individual pharmacophore hypothesis al compounds with the correct number and type of functional groups that were spatialy within a root-mean-square difference (RMSD) < 1.2 Å of the features were considered hits with a maximum of 10,000 being selected. Al hits from the individual pharmacophores for each site were then pooled for further analysis. Table 3. Combination of pharmacophore features used for screening the ces es189 43011.601_P18269-03

[0475] The hit compounds were then subjected to free energy scoring based on the SILCS-MC approach and the obtained ligand grid free energy (LGFE) scores. Calculating LGFE involves classification of the ligand atoms into FragMap type, folowed by the summation of GFEs at the coordinate position of the classified ligand atoms. Ustach et al., 2019; Goel et al., 2021.

[0476] A positive GFE value of 1000 is assigned for atoms faling in forbidden regions defined by the SILCS exclusion map. SILCS-MC involves sampling of the orientation and conformation of the ligand in the field of the FragMaps, based on the LGFE score along with the intramolecular energy of the ligand based on the CGenFF function with a 1 / r dielectric constant. Vanommeslaeghe et al., 2010.

[0477] SILCS-MC pose refinement was performed starting from the docked orientation of each ligand from the pharmacophore search. MC sampling involved 100 steps of translations, molecular rotations, and dihedral rotations of 0.5 Å, 15^ and 180^, respectively folowed by MC simulated annealing of 1000 steps of 0.25 Å, 9^ and 9^, respectively, for a minimum of 50 runs based on a convergence of 0.5 kcal / mol up to a maximum of 250 runs. Five repeats of this protocol were performed at each site and binding pose and orientation of ligands were ranked based on most negative LGFE scores. Final compound selection considered physical properties indicative of drug-likeness, also known as Lipinski rule of five, i.e., logP(o / w), number of H-bond donors and acceptors, and molecular mass were obtained for the selected compounds using MOE (Chemical Computing Group).4-dimensional bioavailability (4DBA) values, Oashi et al., 2011, were also calculated which combines Lipinski’s rule of five, Lipinski, 2000, into a single scalar value. Ligands with molecular weight < 200 and > 550 Daltons were discarded. Ligands that did not pass the PAINS rules, Bael and Holoway, 2010, indicating potential toxicity related issues were also removed. The remaining ligands were grouped into clusters based on chemical similarity metric, i.e., Morgan fingerprinting (cutof = 0.45) with compounds for biological evaluation obtained by choosing ligands with the most negative LGFE scores in each cluster.

[0478] Three putative binding sites for drug-like molecules were identified on HIF-2α, based on SILCS-Hotspots analysis and visual inspection (FIG.14c). Site 1 is located in bHLH domain, exhibiting aliphatic, aromatic, and positively charged group maps with significant hydrogen-bond donor and acceptor maps. Site 2 is a relatively exposed region of β-sheets dominated by apolar and hydrogen bond acceptor maps. Although this site is a key region of contact between HIF-α and HIF-1β subunits of HIF and is dominated by aliphatic and aromatic maps, the flatness of the site 190 43011.601_P18269-03and dominance of hydrophobic interactions make it a poor choice for drug binding. Site 3 located in PAS-B domain is the most interesting site for ligand-binding as it provides a wel-defined pocket dominated by aliphatic, aromatic, hydrogen bond acceptor and positively charged group FragMaps. PAS-B subdomain is where the compound PT2385 binds, Walace et al., 2016, indicating its utility as a potential drug-binding site that wil yield a therapeutic outcome.

[0479] Virtual database screening was performed targeting each site involving first the pharmacophore screening folowed by final ranking based on the SILCS-MC LGFE scores. For each site, the top 1000 ranked ligands from each database were selected for final filtering based on physical properties, including Lipinski’s rule of 5 and the 4DBA metric. From this procedure 100 compounds were selected for each site. Examples of candidate ligands in the predicted binding orientation at each site are shown in FIG.14d.

[0480] Chemical Synthesis

[0481] General procedures. Al commercialy available reagents and solvents were used without further purification unless otherwise stated. Automated flash chromatography was performed on a Teledyne Isco CombiFlash Rf+ using Teledyne Isco flash silica cartridges. Spectra were recorded on a JEOL JNM-LA500 spectrometer (¹H NMR at 500 MHz and ¹³C NMR at 125 MHz) at 295 K in deuterated chloroform (CDCl3) (¹H NMR referenced to internal standard tetramethylsilane 0.00 ppm, ¹³C NMR referenced to 77.00 ppm), deuterated dimethyl sulfoxide (d6-DMSO) (¹H NMR referenced to 2.50 ppm, ¹³C NMR referenced to 39.510 ppm). Analytical LC-MS was performed using Agilent 1260 equipped with autosampler (Agilent Poroshel 120 column (50 × 3.0 mm I.D., 2.7 μm); 0.05% trifluoroacetic acid (TFA) in water / acetonitrile gradient; UV detection at 215 and 254 nm) and electrospray ionization. Unless otherwise noted, al final compounds showed purity greater than 95% at 215 and 254 nm using this method.

[0482] Synthetic procedures

[0484] Ethyl 3-(3-(benzyloxy)cyclobutyl)-3-oxopropanoate: This procedure is a modification of that reported by Yamaguchi-Sasaki et al., 2020. To a solution of 3- 191 43011.601_P18269-03benzyloxycyclobutanecarboxylic acid (4.13 g, 20 mmol) in tetrahydrofuran (THF; 40 mL), 1,1′- carbonyldimidazole (CDI; 3.89 g, 24 mmol) at room temperature was added. The mixture was stired under N2 for 1 hour then mono-methyl malonate potassium salt (5.1110 g, 30 mmol) and MgCl2 (2.29 g, 24 mmol) were added to the above solution. The resulting mixture was stired at room temperature overnight. It was then quenched with aqueous HCl (1 N, 80 mL) and was extracted with ethyl acetate (EtOAc; 3 × 50 mL). The combined organic phases were washed with water (50 mL) and then brine (aqueous saturated NaCl solution; 50 mL × 3), treated with anhydrous sodium sulfate (Na2SO4), filtered, and concentrated under reduced pressure. The residue was purified by flash chromatography to give the desired product (5.19 g, 94%) as a clear liquid.

[0485] ¹H NMR (500 MHz, CDCl3) d 7.71-7.38 (m, 5H), 4.35-4.46 (m,2H), 4.10-4.24 (m, 2H), 3.91-4.05 (m, 2H) 3.37-3.47 (m, 2H), 2.81-2.94 (m, 1H), 2.37-2.61 (m, 2H), 2.08-2.31 (m, 2H), 1.17-1.38 (m, 3H); LC-MS (ESI): tR=2.35 min, m / z calculated for C16H2OO4Na [M+Na]+ : 299.1, found: 299.2.- mL, 27 mmol) in MeOH (15 mL), formamidine acetate (1.41 g, 13.5 mmol) was added in batches for 30 minutes. Cyclobutyl-3-oxopropanoate (2.49 g, 9 mmol) in methanol (MeOH; 15 mL) was then added dropwise. The mixture was stired for 24 hours at room temperature. Water (10 mL) and acetic acid (1.7 ml) were added and the solvent was removed under reduced pressure. The residue was extracted with dichloromethane / MeOH (20 / 1, v / v) (50 mL × 1, then 25 mL × 2). The combined organic layer was dried over sodium sulfate, filered and concentrated. The residue was purified on flash column chromatography (5% MeOH in CH2Cl2) give the desired product (1.78 g, 77.2%) as a white solid.

[0488] ¹H NMR (500 MHz, CDCl3) d 8.24 (br. s., 1H), 7.32-7.40 (m, 5H), 7.27-7.32 (m, 1H), 6.31 (s, 1H), 4.47 (s, 2H), 4.02-4.10 (m, 2H), 2.88-2.98 (m, 1H), 2.58-2.67 (m, 2H), 2.45-2.52 (m, 1H), 192 43011.601_P18269-032.17- 2.27 (m, 2H); LC-MS (ESI): tR=1.92 min, m / z calculated for C15H17N2O2 [M+H]+ : 257.1, found: 257.0.

[0490] 4-(3-(benzyloxy)cyclobutyl)-6-chloropyrimidine: To a solution of 6-(3- (benzyloxy)cyclobutyl) pyrimidin-4(1H)-one (1.02 g, 3.98 mmol) in 1,2-dichloroethane (25 mL), POCl3 (1.86 mL) was added. The reaction mixture was heated at 100°C for 2 hours in a round- botom flask equipped with a refluxing condenser. The resulting mixture was cooled to room temperature, diluted in EtOAc (80 mL) and neutralized with saturated NaHCO3 solution (80 mL). The aqueous phase was extracted with EtOAc (40 mL × 2). The combined organic layers were washed with brine (60 mL × 4), dried over Na2SO4, filtered, and the organic phase was concentrated. The residue was purified on flash column chromatography (15% EtOAc in hexane, 25 g size) to give a cis isomer (0.502 g, 45.9%) as a white solid and a trans isomer (0.132 g, 12.1%) as a white solid.

[0491] cis-4-(3-(benzyloxy)cyclobutyl)-6-chloropyrimidine ¹H NMR (500 MHz, CDCl3) d 8.92 (s, 1H), 7.33-7.39 (m, 4H), 7.27-7.33 (m, 1H), 7.24 (s, 1H), 4.48 (s, 2H), 4.07-4.15 (m, 1H), 3.04- 3.14 (m, 1H), 2.65-2.74 (m, 2H), 2.26-2.35 (m, 2H), ¹³C NMR (126 MHz, CDCl3) d 173.7, 161.2, 158.7, 137.9, 128.4, 127.9, 127.7, 119.2, 70.2, 68.6, 36.3, 32.2; LC-MS (ESI): tR=2.47 min, m / z calcd for C15H16ClN2O [M+H]+ : 275.1, found: 275.0.

[0492] trans-4-(3-(benzyloxy)cyclobutyl)-6-chloropyrimidine ¹H NMR (500 MHz, CDCl3) d 8.92 (s, 1H), 7.32-7.37 (m, 4H), 7.26-7.32 (m, 1H), 7.20 (s, 1H), 4.46 (s, 2H), 4.37-4.44 (m, 1H), 3.53- 3.61 (m, 1H), 2.48-2.59 (m, 4H), ¹³C NMR (126 MHz, CDCl3) d 175.1, 161.1, 158.9, 138.1, 128.4, 127.8, 127.7, 119.6, 71.4, 70.3, 35.2, 34.8; LC-MS (ESI): tR=2.50 min, m / z calculated for C15H16ClN2O [M+H]+ : 275.1, found: 275.0. 193 43011.601_P18269-03

[0493]

[0494] tert-butyl (2-(2-ethylphenoxy)ethyl)(methyl)carbamate: To a solution of 2- ethylphenol (2.44 g, 20 mmol), tert-butyl (2-hydroxyethyl) (methyl)carbamate (3.86 g, 22 mmol) in anhydrous THF (100 mL), triphenylphosphine (7.87 g, 30 mmol) and a solution of DIAD (disopropyl azodiformate) (6.07 g, 30 mmol) in anhydrous THF (30 mL) was added at room temperature. The mixture was stired under an argon atmosphere at room temperature overnight. The mixture was concentrated and the resulting residue was redissolved in EtOAc (150 mL). The organic layer was washed with saturated NaHCO3 (50 mL) and washed with saline (50 mL × 3). The organic layer was dried with sodium sulfate (anhydrous), filtered and concentrated. The concentrate was partialy purified by silica-gel column chromatography to give the desired product (2.88 g, 51.5%) as a clear liquid along with 2-ethylphenol (about 30-50%).

[0495] 1H NMR (500 MHz, CDCl3 d 7.11-7.19 (m, 2H), 6.86-6.95 (m, 1H), 6.81 (d, J = 7.9 Hz, 1H), 4.02- 4.15 (br, m., 2H), 3.56-3.70 (br. m., 2H), 3.01 (s, 3H), 2.64 (q, 7.5 Hz, 2H), 1.47 (s, 9H), 1.19 (t, 7.5 Hz, 3H); LC-MS (ESI): tR=2.85 min, m / z calcd for C15H25NO3Na [M+Na]+ : 302.2, found: 302.1.

[0497] 2-(2-ethylphenoxy)-N-methylethan-1-aminium chloride: To a solution of tert-butyl (2- (2- ethylphenoxy)ethyl)(methyl)carbamate (2.88 g, 10.3 mmol) in dichloromethane (52 mL) was added a solution of HCl (26 mL, 4 M in 1,4-dioxane). The mixture was stired at13 room temperature overnight. The solvent (dichloromethane) was removed under reduced pressure. The mixture was then precipitated and washed with ether to give the desired product (1.28 g, 57.4%) as a white salt. 194 43011.601_P18269-03

[0498] 1H NMR (500 MHz, DMSO-d6) d 9.06 (br. s., 2H), 7.15-7.21 (m, 2H), 6.95-6.99 (m, 1H), 6.93 (td, J = 7.4 Hz, 0.9 Hz, 1H), 4.26 (t, J = 5.2 Hz, 2H), 3.28-3.38 (m, 2H), 2.65 (s, 3H), 2.61- 2.68 (m, 2H), 1.15 (t, J = 7.5 Hz, 3H);13C NMR (125.7 MHz, DMSO-d6) d 155.4, 132.0, 128.8, 126.9, 121.1, 111.6, 63.4, 47.3, 32.9, 22.3, 14.3; LC-MS (ESI): tR=1.72 min, m / z calcd for C11H18NO [M- Cl]+ :180.1, found: 179.9.

[0500] Cis-6-(3-(benzyloxy)cyclobutyl)-N-(2-(2-ethylphenoxy)ethyl)-N-methylpyrimidin-4- amine: To a solution of cis-4-(3-(benzyloxy)cyclobutyl)-6-chloropyrimidine (560 mg, 2.04 mmol) in ethanol (20 mL), 2-(2-ethylphenoxy)-N-methylethan-1-aminium chloride (572 mg, 2.65 mmol) and triethylamine (852 μL, 6.11 mmol) was added. The reaction mixture was heated at 90°C in a sealed tube for 40 hours. The mixture was then concentrated under reduced pressure. The residue was purified on flash column chromatography (90% EtOAc / 10% EtOH as bufer A; Hexane as bufer B) to give the desired product (0.850 g, 99.9%) as a slightly yelow solid.

[0501] 1H NMR (500 MHz, CDCl3) d 8.56 (s, 1H), 7.20-7.40 (m, 5H), 7.05-7.20 (m, 2H), 6.86- 6.95 (m, 1H), 6.8 (d, J = 8.3 Hz, 1H), 6.32 (s, 1H), 4.47 (s, 2H), 4.13-4.25 (m, 2H), 3.92-4.13 (m, 3H), 3.19 (s, 3H), 2.86-3.01 (m, 1H), 2.61-2.72 (m, 2H), 2.50-2.61 (m, 2H), 2.16-2.33 (m, 2H), 1.14 (t, J = 7.3 Hz, 3H);13C NMR (125.7 MHz, CDCl3) d 169.7, 161.9, 157.8, 156.3, 138.2, 132.4, 129.0, 128.4, 127.9, 127.6, 126.8, 120.7, 110.6, 99.1, 70.0, 68.8, 49.1, 35.9, 32.3, 23.3, 14.1;13C NMR (125.7 MHz, DMSO-d6) d 161.7, 157.5, 156.0, 138.5, 131.6, 129.8, 128.8, 128.2, 127.7, 127.4, 126.9, 120.5, 111.0, 99.9, 69.0, 68.4, 65.2, 60.0, 48.0, 35.5, 31.7, 22.8, 14.2; LC-MS (ESI): tR=2.57 min, m / z calculated for C26H32N3O2 [M+H]+ :418.3, found: 418.4. 195 43011.601_P18269-03

[0502]

[0503] Cis-3-(6-(2-(2-(3.2.16): To a solution of cis-6-(3-(benzyloxy)cyclobutyl)-N-(2-(2-ethylphenoxy)ethyl)-N- methylpyrimidin-4-amine (320 mg, 0.60 mmol) in ethanol (32 mL), Pd(OH)2 (320 mg) was added. The mixture was stired under hydrogen using a baloon for 6-8 days. The resulting mixture was filtered through celite. The filtrate was concentrated and the residue was purified on flash column chromatography (dichloromethane / methanol) to give the desired product (95 mg, 38%).

[0504] 1H NMR (500 MHz, CDCl3) d 8.59 (s, 1H), 7.11-7.18 (m, 2H), 6.88-6.93 (m, 1H), 6.8 (d, J = 8.3 Hz, 1H), 6.32 (br. s., 1H), 4.24-4.31(m, 1H), 4.19 (t, J = 5.2 Hz, 2H), 3.96-4.14 (m, 2H), 3.22 (s, 3H), 3.04 (q, J = 7.6 Hz, 1H), 2.75-2.84 (m, 2H), 2.54-2.62 (m, 2H), 2.16-2.26 (m, 2H), 1.13 (t, J = 7.4 Hz, 3H);13C NMR (125.7 MHz, CDCl3) d 169.8, 161.9, 157.7, 156.2, 132.3, 129.0, 126.8, 120.8, 110.6, 99.9, 64.8, 49.2, 38.7, 34.3, 23.3, 14.1; LC-MS (ESI): tR=2.09 min, m / z calcd for C19H26N3O2 [M+H]+ :328.2, found: 328.2.a solution of cis-4-(3-(benzyloxy)cyclobutyl)-6-chloropyrimidine (103 mg, 0.37 mmol) in ethanol (20.0 mL), 1-[2-(Phenoxy)-ethyl]piperazine (155 mg, 0.75 mmol) was added. The reaction mixture was heated at 90°C in a sealed tube for 16 hours. The mixture was cooled to room temperature and concentrated under reduced pressure. The residue was purified on flash column chromatography (CH2Cl2 as Bufer A; 95% MeOH and 5% ammonia water (30%), v / v as Bufer B) to give the desired product (150 mg, 90.2%).1H NMR (500 MHz, CDCl3) d 8.55 (s, 1H), 7.27-7.41 (m, 7H), 6.85-7.01 (m, 3H), 6.36 (s, 1H), 4.47 (s, 2H), 4.11-4.21 (m, 2H), 4.02-4.11 (m, 1H), 3.68 (br. m., 4H), 2.89-3.00 (m, 1H), 2.82-2.89 (m, 2H), 2.17-2.30 (m, 2H);13C NMR (125.7 MHz, CDCl3) d 170.1, 162.0, 158.6, 158.1, 138.1, 129.5, 128.4, 127.9, 127.6, 120.9, 114.5, 99.2, 70.0, 68.8, 65.8, 196 43011.601_P18269-0357.2, 53.1, 43.7, 36.0, 32.4; LC-MS (ESI): tR= 2.06 min, m / z calcd for C27H33N4O2 [M+H]+ : 445.3, found: 445.6.a solution of cis-4-(3-(benzyloxy)cyclobutyl)-6-(4-(2-phenoxyethyl)piperazin-1- yl)pyrimidine (70 mg, 0.16 mmol) in ethanol (7.0 mL), Pd(OH)2 (70 mg) was added. The mixture was stired under hydrogen using baloon for 6-8 days The resulting mixture was filtered through celite. The filtrate was concentrated and the residue was purified on flash column chromatography (CH2Cl2 as Bufer A; 95% MeOH and 5% ammonia water (30%), v / v as Bufer B) to give the desired product (16 mg, 28%).

[0509] 1H NMR (500 MHz, CDCl3) d 8.58 (s, 1H), 7.27-7.38 (m, 2H), 6.86-7.00 (m, 3H), 6.33 (s, 1H), 4.23-4.32 (m, 1H), 4.09-4.19 (m, 2H), 3.61-3.76 (br. m., 4H), 2.97-3.08 (m, 1H), 2.82-2.91 (m, 2H), 2.73-2.82 (m, 2H), 2.59-2.71 (m, 4H), 2.07-2.17 (m, 2H);13C NMR (125.7 MHz, CDCl3) δ 161.8, 158.6, 158.0, 129.5, 120.9, 114.5, 99.9, 77.3, 65.7, 64.8, 57.2, 53.1, 43.7, 38.7, 34.3; LC- MS (ESI): tR= 1.10 min, m / z calcd for C20H27N4O2 [M+H]+ : 355.2, found: 355.3.

[0510]

[0511] of isothiocyanatobenzene (568 mg, 4.20 mmol) in methylene chloride (20 mL) at room temperature was added 1,3-di(piperidinyl)propane (421 mg, 2 mmol) in portion wise. The reaction mixture was stired at room temperature for 2 hours (the product was precipitated). The precipitated solids were filtered washing with dichloromethane (20 mL) and the filtrate was purified on flash column chromatography (5% MeOH in dichloromethane) to give the desired product (858 mg, 89.2%) as a white solid.1H NMR (500 MHz, DMSO-d6) d 9.19 (s, 2H), 7.21- 7.31 (m, 8H), 7.04-7.13 (m, 2H), 4.64-4.78 (m, 4H), 3.02 (t, J = 12.7 Hz, 4H), 1.66-1.78 (m, 4H), 197 43011.601_P18269-031.49-1.62 (m, 2H), 1.29-1.40 (m, 2H), 1.19-1.29 (m, 4H), 1.12 (q, J = 11.9 Hz, 4H) ;13C NMR (125.7 MHz, DMSO-d6) δ 180.7, 141.3, 127.9, 125.1, 124.0, 48.5, 35.9, 35.1, 31.8, 23.2; LC-MS (ESI): tR= 2.76 min, m / z calculated for C27H40N4S2 [M+O+H]+ : 497.2, found:497.6, [M+O+2H]2+: 249.13, found: 249.0.: a g, mmol, freshly made) in methylene chloride (10 mL) at room temperature was added 1,3- di(piperidinyl)propane (183 mg, 0.87 mmol). The reaction mixture was stired for 2 hours. The reaction mixture was concentrated under reduced pressure and the residue was purified on flash column chromatography (10% MeOH in methylene chloride) to give the desired product (405 mg, 91.1%) as an orange solid.

[0514] 1H NMR (500 MHz, DMSO-d6) d 8.51 (m, 4H), 8.25 (t, J = 5.0 Hz, 2H), 7.21-7.39 (m, 4H), 4.55- 4.74 (m, 4H), 2.98 (t, J = 12.2 Hz, 4H), 1.61-1.79 (m, 4H), 1.45-1.60 (m, 2H), 1.28- 1.39 (m, 2H), 1.13-1.27 (m, 4H), 0.95-1.13 (m, 4H);13C NMR (125.7 MHz, CDCl3) δ 181.1, 149.3, 149.1, 122.0, 47.9, 47.3, 35.9, 35.2, 31.7, 23.1; LC-MS (ESI): tR= 2.05 min, m / z calculated for C19H28N3O228 [M+2H]2+: 256.1, found: 256.1; [M+1H]+ : 511.3, found: 511.5.17. Table 4. Cancer cel lines used in this Example. 1 +198 43011.601_P18269-03Table 4. Cancer cel lines used in this Example. Cel line Catalog Source Complete medium*# % % S199 43011.601_P18269-03Table 4. Cancer cel lines used in this Example. Cel line Catalog Source Complete medium*# 5 S % + m; S,200 43011.601_P18269-03Table 5. Antibodies against human (h) or mouse (m) proteins used for immunoblot assays (IB), immunohistochemistry (IHC), immunoprecipitation ne201 43011.601_P18269-03Table 5. Antibodies against human (h) or mouse (m) proteins used for immunoblot assays (IB), immunohistochemistry (IHC), immunoprecipitation ne202 43011.601_P18269-03Table 5. Antibodies against human (h) or mouse (m) proteins used for immunoblot assays (IB), immunohistochemistry (IHC), immunoprecipitation ne203 43011.601_P18269-03Table 5. Antibodies against human (h) or mouse (m) proteins used for immunoblot assays (IB), immunohistochemistry (IHC), immunoprecipitation neTable 6. PCR primer pairs (forward [F] and reverse [R]) used for analysis of human (H) and n204 43011.601_P18269-03Table 6. PCR primer pairs (forward [F] and reverse [R]) used for analysis of human (H) and mouse (m) mRNA levels in cel lines and tumor tissue. n205 43011.601_P18269-03Table 6. PCR primer pairs (forward [F] and reverse [R]) used for analysis of human (H) and mouse (m) mRNA levels in cel lines and tumor tissue. n206 43011.601_P18269-03Table 6. PCR primer pairs (forward [F] and reverse [R]) used for analysis of human (H) and mouse (m) mRNA levels in cel lines and tumor tissue. n207 43011.601_P18269-03Table 6. PCR primer pairs (forward [F] and reverse [R]) used for analysis of human (H) and mouse (m) mRNA levels in cel lines and tumor tissue. nSupplementary Table 7. Cancer cel lines and mouse strains used for tumor studies. u, u,208 43011.601_P18269-03Supplementary Table 7. Cancer cel lines and mouse strains used for tumor studies. Cel line Injected Site Mouse strain sex e ld, eSupplementary Table 8. Formulation of drugs administered in vivo.209 43011.601_P18269-03Supplementary Table 8. Formulation of drugs administered in vivo. Formulation for IP administrationSupplementary Table 9. Antibodies used for flow cytometry (FC).210 43011.601_P18269-03Supplementary Table 9. Antibodies used for flow cytometry (FC). Antibodies Catalog number Source Application*inin;EXAMPLE 3 Compounds 3.2n and 3.2.16n

[0515] Two other compounds that were tested in vivo were 3.2n and 3.2.16n, which are derived from 3.2 and 3.2.16, respectively, by elimination of the hydroxyl group on the cyclobutyl moiety: 211 43011.601_P18269-036n). t cancer in which HCC1954 human breast cancer cels were orthotopicaly implanted into the mammary fat pad of immunodeficient mice and treated with the compounds at the indicated doses for 7 days once the tumors reached a volume of 150 mm3 (FIG.19A-FIG.19C). Compound 3.2n also was active in the HCT116 human colorectal cancer xenograft model (FIG.20A-FIG.20D) and the orthotopic EMT6 mouse triple-negative breast cancer model in syngeneic and immunocompetent Balb / c mice (FIG.21A-FIG.21H). EXAMPLE 4 Compounds of formula (I) and formula (I)

[0518] Representative compounds of formula (I) and formula (I) are provided in Tables 10 and 11, respectively, along with physical characteristics and assay data. SILCS IC50 values of representative compounds of formula (I) and formula (I) against various HIF targets are provided in Table 12. 212 43011.601_P18269-03sdnuop mo C det 2 2. n n 4. 5. 8. 3. 9. 4. 8. n 3. . 6. n n 8. 8. nc . 7 2 4 2 0 0 5 0 1 0e 1 1 5 0 0leS T r 9 9 G P 9 9 9 9 9 9 AI 9 AI 9 9 9 G AIo F f I A A R C C D G A A A A A A F A F A F G A A R F N C C C C C C P C P C P C C D P s H N P P N Pe A ul -a B 9 9 -9 1 45 6 V o M - 41 51 5 7- D . 4 5 7 9 1 9 1 2 -0 mo - A 1 F M M C 74 7 4- 1 1 C T -T D 5 CI H A D D M U S U M T R T S U C a C H L 78 1 B3 u 2 S M Z B Le H D 52 p D 6 a 5S M H U e t H U H F KC b B C + R R R M M SLI u S N E E E C C M S T H . C2 l t 1 C ts ac ce a Ce L l I a iv r l m r o ev CSb S er r ol a il ia B e o T C L N C G H 213 43011.601_P18269-03. . 1. 6 7 5 9 1 . 8 0 0. . . . . . 0 . 0 0 4 0 2 1 < 0 2. 4. 4. 2. s n s n 1. 4. 5. 9 s s s s 4 . 6 3 0 8 3 4 0 1 .1 n n n n 2. 8. 9. 2. 7. 2. 9. 6. 2. s s 0 0 0 8 1 0 0 2 0 n n s n s n 5. 0 6 s s 0 s s s s 0 .2 .7 n n 1 1. 8. 3. 2. n n n n > 5 5 9 6 T T A A 9 9 P P 9 9 AI TP F F T T A A C C G G A A F 9 9 A A M N N C C P C C D G G G U E E L UL A A P A N A V V G G 5 s- 3 P - a B2 4 2 1 3 2 9 5 R 3 3 1 C - C 8 0 4 L 4 7 - - 3 A V C N 4 C U P O D - 9 1 C C 5 - C O C L 6 4- C C K A A V - Px N 87 A C R O K S B A P R R e R A A dn i . p i i me g mo n t ai a e e ta l t a k n n r r n u u al a c c s n o e e L e v r L M O a P R P 214 43011.601_P18269-03REFERENCES

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[0622] Although the foregoing subject mater has been described in some detail by way of ilustration and example for purposes of clarity of understanding, it wil be understood by those skiled in the art that certain changes and modifications can be practiced within the scope of the appended claims. 224 43011.601_P18269-03

Claims

THAT WHICH IS CLAIMED:

1. A compound of formula (I): ; wherein:is a can or L1 is a linker selected from –(CH2)t-, -O-CH2-CH2-, and -CH2-CH2-O-; wherein t is an integer selected from 1, 2, and 3; A1 and A2 are each independently -CH- or N; B1 and B2 can be present or absent and when present are each independently selected from -CR1R2- and -C(C=O)-, wherein R1 and R2 are each independently H or C1-C4 alkyl; X1 and X2 are each independently selected from S, O, and SR3, wherein R3 is C1-C4 alkyl; Y1 and Y2 are each independently selected from -CH2-, -NR4-, and -O-, wherein R4 is C1- C4 alkyl, or Y1 is -N- when X1 is -SR3 and / or Y2 is -N- when X2 is -SR3. Z1 and Z2 are each independently selected from C1-C4 alkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl; and stereoisomers and pharmaceuticaly acceptable salts thereof.

2. The compound of claim 1, wherein L1 is -(CH2)3- and the compound of formula (I) is a compound of formula (Ia): .

3. The compound of claim 2, wherein the compound of formula (Ia) is selected from:225 43011.601_P18269-03-i).

4. The compound of claim 3, wherein X1 and X2 are each independently S or O and the compound of formula (Ia-i) and compound of formula (Ia-i) are: .

5. The compound of claim 4, wherein B is absent and the compound of formula (Ia- a) and compound of formula (Ia-i) are: .

6. The compound of any one of claims 1 to 5, wherein Z1 and Z2 are each independently selected from substituted or unsubstituted C1-C8 straight chain or branched alkyl, phenyl, pyrimidinyl, imidazoyl, pyrazinyl, and pyridazinyl.

7. The compound of claim 6, wherein Z1 and Z2 are each independently selected from: 226 43011.601_P18269-03; w n is an integer selected from 0, 1, 2, 3, 4, and 5; m is an integer selected from 0, 1, 2, 3, and 4; p is an integer selected from 0, 1, 2 and 3; and q is an integer selected from 0, 1, and 2; and each R5 is independently selected from halogen, C1-C4 alkyl, hydroxyl, alkoxyl, cyano, - CF3, -NO2, -C(=O)H, -SR6, -SO2R7, -C(=O)-R8, and -C(=O)-O-R9, wherein R6, R7, R8, and R9 are independently C1-C4 alkyl.

8. The compound of any one of claims 1 to 7, wherein the compound of formula (I) is selected from a compound listed in Table 10.

9. The compound of any one of claims 1 to 8, wherein the compound of formula (I) is selected from:227 43011.601_P18269-0310. A compound of formula (I): (I); wherein:L2 is a linker which can be present or absent and when present is selected from: ; wherein selectedfrom 0, 1, 2, and 3, each X3 can be present or absent and when present is -O-, and R13 is selected from H, C1-C4 alkyl, and -NR14R15, wherein R14 and R15 are each H or C1-C4 alkyl; R10 is selected from a substituted or unsubstituted cycloalkyl or heterocycloalkyl, straight chain or branched C1-C4 alkyl, C1-C4 alkoxyl, -C(=O)-O-R16, and -CH2-C(=O)-O-R17, wherein R16 and R17 are each independently C1-C4 alkyl; R11 is selected from H, straight chain or branched C1-C4 alkyl, -NR18R19, wherein R18 and R19 are each H or C1-C4 alkyl; R12 is selected from H and halogen; or R10 and R12 together form a 5- to 6- membered cycloalkyl or heterocycloalkyl ring; Z3 is selected from C1-C4 alkyl, aryl, and heteroaryl, each of which can be substituted or unsubstituted; and stereoisomers and pharmaceuticaly acceptable salts thereo.

11. The compound of claim 10, wherein ring C is selected from: .228 43011.601_P18269-0312. The compound of claim 10 or claim 11, wherein the compound of formula (I) is selected from:

13. The compound of any one of claims 10 to 12, wherein R10 is a substituted or unsubstituted cycloalkyl or heterocycloalkyl selected from: ; wherein:R20 is selected from H, C1-C4 alkyl, hydroxyl, and -O-C(=O)-R24, wherein R24 is C1-C4 alkyl; R21 is H or C1-C4 alkyl; R22 is selected from H, C1-C4 alkyl, and phenyl; or R20 and R21 together form a 1,3-dioxolane ring structure.

14. The compound of any one of claims 10 to 13, wherein Z3 is selected from: 229 43011.601_P18269-03N (R25)n3 N N (R25)m1 (R25)m1 (R25)m1 ;n3 is an integer selected from 0, 1, 2, 3, 4, and 5; m1 is an integer selected from 0, 1, 2, 3, and 4; p1 is an integer selected from 0, 1, 2 and 3; and q1 is an integer selected from 0, 1, and 2; and each R25 is independently selected from halogen, C1-C4 alkyl, hydroxyl, alkoxyl, cyano, - CF3, -NO2, -C(=O)H, -SR26, -SO2R27, -C(=O)-R28, and -C(=O)-O-R29, wherein R26, R27, R28, and R29 are independently C1-C4 alkyl.

15. The compound of any one of claims 10 to 14, wherein the compound of formula (I) is selected from a compound listed in Table 11.

16. The compound of any one of claims 10 to 15, wherein the compound of formula (I) is selected from: 230 43011.601_P18269-03.2).

17. A formulation comprising a compound of formula (I) or formula (I) of any one of claims 1 to 16 and an pharmaceuticaly acceptable carier.

18. The formulation of claim 17, wherein the formulation comprises a liposomal composition, niosomes, nanoemulsions, nanosuspensions, miceles, nanomiceles, hydrogels, polymeric nanoparticles or microparticles, including a nanoparticle or microparticle comprising a poly(lactic-co-glycolic)acid (PLGA), a poly(ε-caprolactone) (PCL), a poly(lactic acid) (PLA), a poly(glycolic acid) (PGA), a polyester, a poly(orthoester), a poly(phosphazine), a poly(phosphate ester), a poly(ε-caprolactone-co-ethyl ethylene phosphate) (PCLEEP), a polyvinyl alcohol (PVA), a poly(β-amino ester), a poly(acrylic acid) (PAA), a poly-3-hydroxybutyrate (P3HB), a poly(hydroxybutyrate-co-hydroxyvalerate), a polyethylene glycol (PEG), and combinations thereof, solid lipid nanoparticles, nanostructured lipid cariers, nanocrystals, dendrimers, cubosomes, olaminosomes, bilosome, extracelular vesicles (exosomes), and smart nano-micro platforms.

19. A method for treating a disease, disorder, or condition associated with one or more hypoxia inducible factors (HIFs) in a subject in need of treatment thereof, the method comprising administering to the subject a therapeuticaly efective amount of a compound of formula (I) or formula (I) of any one of claims 1 to 16 or the formulation of claim 17 or claim 18.

20. The method of claim 19, wherein the disease, disorder, or condition is a cancer. 231 43011.601_P18269-0321. The method of claim 20, wherein the cancer is selected from acute lymphoblastic leukemia (ALL), acute myeloid leukemia, adrenocortical carcinoma, adult acute, AIDS-related cancers, AIDS-related lymphoma, anal cancer, appendix cancer, arcoma, astrocytoma (childhood cerebelar or cerebral), barcinoid tumor, basal-cel carcinoma, bile duct cancer, bladder cancer bone tumor, brain cancer, brain tumor, brainstem glioma, breast cancer, bronchial adenomas / carcinoids, Burkit’s lymphoma, carcinoma of unknown primary, cerebelar astrocytoma, cerebral astrocytoma / malignant glioma, cervical cancer, childhood, childhood acute, childhood cerebral astrocytoma, childhood visual pathway and hypothalamic, chondrosarcoma, chronic, chronic lymphocytic, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloid leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous, cutaneous t-cel, cutaneous t-cel lymphoma, desmoplastic smal round cel tumor, endometrial, endometrial cancer, ependymoma, esophageal cancer, Ewing family of tumors, Ewing’s sarcoma, extracranial germ cel tumor, extragonadal germ cel tumor, extrahepatic bile duct cancer, extrahepatic cancer, galbladder cancer, gastric (stomach) cancer, gastric carcinoid, gastrointestinal, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (gist), germ cel tumor: extracranial, extragonadal, or ovarian, gestational, gestational trophoblastic tumor, glioma, glioma of the brain stem, hairy cel leukemia, hepatocelular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma, intraocular melanoma, islet cel carcinoma (endocrine pancreas), Kaposi sarcoma, kidney cancer (renal cel cancer), laryngeal cancer, leukaemias, lip and oral cavity cancer, liposarcoma, liver cancer (primary), lung cancer, lymphomas, macroglobulinemia, malignant fibrous histiocytoma of bone / osteosarcoma, meduloblastoma, melanoma, Merkel cel cancer, mesothelioma, metastatic, mouth cancer, multiple (cancer of the bone-marow), multiple endocrine neoplasia syndrome, multiple myeloma / plasma cel neoplasm, mycosis fungoides, myelodysplastic syndromes, myelodysplastic / myeloproliferative diseases, myelogenous leukemia, myeloid leukemia, myeloma, myeloproliferative disorders, myxoma, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma neuroblastoma, non-Hodgkin lymphoma, non-smal cel, non-smal cel lung cancer, oligodendroglioma, oral cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma, ovarian cancer, ovarian epithelial cancer (surface epithelial-stromal tumor), ovarian germ cel tumor, ovarian low malignant potential tumor, pancreatic cancer, pancreatic 232 43011.601_P18269-03cancer, islet cel, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary adenoma, plasma cel neoplasia / multiple myeloma, pleuropulmonary blastoma, primary central nervous system, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cel carcinoma, renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sezary syndrome, skin cancer (melanoma), skin cancer (non-melanoma), skin carcinoma, smal cel, smal cel lung cancer, smal intestine cancer, soft tissue, soft tissue sarcoma, squamous cel carcinoma, squamous neck cancer with occult primary, stomach cancer, supratentorial primitive neuroectodermal tumors, t-cel lymphoma, testicular cancer, throat cancer, thymoma, thymoma and thymic carcinoma, thyroid cancer, transitional cel cancer, transitional cel cancer of the renal pelvis and ureter, trophoblastic tumor, unknown primary site, ureter and renal pelvis, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma, vulvar cancer, Waldenstrom, Waldenstrom macroglobulinemia, and Wilms tumor (kidney cancer).

22. The method of claim 20, wherein the cancer is selected from breast cancer, colorectal cancer, lung cancer, melanoma, pancreatic cancer, prostate cancer, and head and neck squamous cel carcinoma (HNSCC).

23. The method of claim 20, wherein administration of the compound of formula (I) or formula (I) inhibits or blocks growth and / or vascularization of a tumor associated with breast cancer, colorectal cancer, lung cancer, melanoma, pancreatic cancer, or prostate cancer.

24. The method of claim 19, further comprising administering the compound of formula (I) or formula (I) in combination with an immunotherapy.

25. The method of claim 24, wherein the immunotherapy comprises immune checkpoint blockade (ICB) immunotherapy.

26. The method of claim 25, wherein the ICB immunotherapy includes anti-CTLA-4, anti-PD-1, or anti-PD-L1 immunotherapy. 233 43011.601_P18269-0327. The method of any one of claim 24 to claim 26, wherein administering the compound of formula (I) or formula (I) in combination with the immunotherapy alters an immune cel microenvironment of a tumor associated with acute lymphoblastic leukemia (ALL), acute myeloid leukemia, adrenocortical carcinoma, adult acute, AIDS-related cancers, AIDS-related lymphoma, anal cancer, appendix cancer, arcoma, astrocytoma (childhood cerebelar or cerebral), barcinoid tumor, basal-cel carcinoma, bile duct cancer, bladder cancer bone tumor, brain cancer, brain tumor, brainstem glioma, breast cancer, bronchial adenomas / carcinoids, Burkit’s lymphoma, carcinoma of unknown primary, cerebelar astrocytoma, cerebral astrocytoma / malignant glioma, cervical cancer, childhood, childhood acute, childhood cerebral astrocytoma, childhood visual pathway and hypothalamic, chondrosarcoma, chronic, chronic lymphocytic, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloid leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous, cutaneous t-cel, cutaneous t-cel lymphoma, desmoplastic smal round cel tumor, endometrial, endometrial cancer, ependymoma, esophageal cancer, Ewing family of tumors, Ewing’s sarcoma, extracranial germ cel tumor, extragonadal germ cel tumor, extrahepatic bile duct cancer, extrahepatic cancer, galbladder cancer, gastric (stomach) cancer, gastric carcinoid, gastrointestinal, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (gist), germ cel tumor: extracranial, extragonadal, or ovarian, gestational, gestational trophoblastic tumor, glioma, glioma of the brain stem, hairy cel leukemia, hepatocelular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma, intraocular melanoma, islet cel carcinoma (endocrine pancreas), Kaposi sarcoma, kidney cancer (renal cel cancer), laryngeal cancer, leukaemias, lip and oral cavity cancer, liposarcoma, liver cancer (primary), lung cancer, lymphomas, macroglobulinemia, malignant fibrous histiocytoma of bone / osteosarcoma, meduloblastoma, melanoma, Merkel cel cancer, mesothelioma, metastatic, mouth cancer, multiple (cancer of the bone-marow), multiple endocrine neoplasia syndrome, multiple myeloma / plasma cel neoplasm, mycosis fungoides, myelodysplastic syndromes, myelodysplastic / myeloproliferative diseases, myelogenous leukemia, myeloid leukemia, myeloma, myeloproliferative disorders, myxoma, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma neuroblastoma, non-Hodgkin lymphoma, non-smal cel, non-smal cel lung cancer, oligodendroglioma, oral cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma, ovarian cancer, ovarian 234 43011.601_P18269-03epithelial cancer (surface epithelial-stromal tumor), ovarian germ cel tumor, ovarian low malignant potential tumor, pancreatic cancer, pancreatic cancer, islet cel, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary adenoma, plasma cel neoplasia / multiple myeloma, pleuropulmonary blastoma, primary central nervous system, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cel carcinoma, renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sezary syndrome, skin cancer (melanoma), skin cancer (non-melanoma), skin carcinoma, smal cel, smal cel lung cancer, smal intestine cancer, soft tissue, soft tissue sarcoma, squamous cel carcinoma, squamous neck cancer with occult primary, stomach cancer, supratentorial primitive neuroectodermal tumors, t-cel lymphoma, testicular cancer, throat cancer, thymoma, thymoma and thymic carcinoma, thyroid cancer, transitional cel cancer, transitional cel cancer of the renal pelvis and ureter, trophoblastic tumor, unknown primary site, ureter and renal pelvis, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma, vulvar cancer, Waldenstrom, Waldenstrom macroglobulinemia, and Wilms tumor (kidney cancer).

28. The method of any one of claim 24 to claim 27, wherein administering the compound of formula (I) or formula (I) in combination with the immunotherapy alters an immune cel microenvironment of a tumor associated with breast cancer, colorectal cancer, lung cancer, melanoma, pancreatic cancer, prostate cancer, or head and neck squamous cel carcinoma (HNSCC).

29. The method of any one of claim 24 to claim 28, wherein administering the compound of formula (I) or formula (I) in combination with an immunotherapy reduces a risk of an adverse event associated with the immunotherapy.

30. The method of claim 19, wherein the disease, disorder, or condition is associated with ocular neovascularization. 235 43011.601_P18269-0331. The method of claim 30, wherein the disease, disorder, or condition associated with ocular neovascularization is selected from diabetic macular edema, diabetic retinopathy, retinal vein occlusion, sickle cel retinopathy, retinopathy of prematurity, Norrie’s disease, Coat’s disease, corneal neovascularization, and age-related macular degeneration.

32. The method of claim 19, wherein administration of the compound of formula (I) prevents expression of VEGF and ANGPTL4.

33. The method of any of claims 19 to 32, further comprising administering one or more additional therapeutic agents.

34. The method of claim 33, wherein the one or more additional therapeutic agents include an anti-cancer agent.

35. The method of claim 34, wherein the anti-cancer agent is selected from an angiogenesis inhibitor, an angiopoietin 2 inhibitor, a CD73 inhibitor, a cyclin dependent kinase inhibitor, an MAP kinase inhibitor, a mTOR inhibitor, a phosphatidylinositol 3-kinase inhibitor, a proteasome inhibitor, a protein phosphatase 2A activator, a serine / threonine kinase inhibitor, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a VEGF-A inhibitor, and a cytotoxic chemotherapy.

36. The method of claim 35, wherein the cytotoxic chemotherapy is selected from 5- fluorouracil, adriamycin, carboplatin, cisplatin, doxorubicin, gemcitabine, idarubicin, and paclitaxel.

37. The method of claim 33, wherein the one or more additional therapeutic agents include an agent for treating a disease, disorder, or condition associated with ocular neovascularization. 236 43011.601_P18269-0338. The method of claim 37, wherein the one or more additional therapeutic agents for treating a disease, disorder, or condition associated with ocular neovascularization include an anti-VEGF agent.

39. The method of claim 38, wherein the anti-VEGF agent is aflibercept, bevacizumab, faricimab, or ranubizumab.

40. The method of any one of claims 19 to 39, wherein the compound of formula (I) or formula (II) is administered prophylacticaly to prevent or reduce an incidence, recurence, or progression of the disease, disorder, or condition.

41. The method of any one of claims 19 to 40, wherein the compound of formula (I) or formula (II) is administered oraly for treating cancer or via intravitreal injection for ocular therapy.

42. The method of any one of claims 19 to 41, wherein the compound of formula(I) or formula (I): (a) directly binds the bHLH domain or the PAS-B subdomain of HIF-1α and HIF-2α; (b) disrupts dimerization with HIF-1β; (c) induces degradation of HIF-1α and HIF-2α; and (d) inhibits HIF target gene expression in one or more cels of the subject. 237 43011.601_P18269-03

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