Compounds containing organometalic stabilized gold (III) macrocycles

Abstract

The presently-disclosed subject matter provides a series of gold (III) macrocyclic compounds, compositions comprising such compounds, methods of making such compounds, and methods of using such compounds.

Description

COMPOUNDS CONTAINING ORGANOMETALIC STABILIZED GOLD (III) MACROCYCLESRELATED APPLICATIONS

[0001] This application claims priority from U.S. Provisional Application Serial No. 63 / 530,400 filed August 2. 2023, the entire disclosure of which is incorporated herein by this reference.GOVERNMENT INTEREST

[0002] This invention was made with government support under grant number R01CA258421-01 awarded by the National Institutes of Health. The government has certain rights in the invention.TECHNICAL FIELD

[0003] The presently-disclosed subject matter relates to compounds and compositions having a unique group of gold (III) complexes stabilized by a C-Au bond and bulky (R,R)- DACH-phenyl Trost ligand via strong o donation. The presently-disclosed subject matter also relates to methods of treating cancer and / or modulating mitochondrial respiration and metabolism using compounds and compositions disclosed herein. The presently-disclosed subject matter also relates to methods of making the compounds and compositions as disclosed herein.INTRODUCTION

[0004] Ovarian cancer (OC), which is characterized as an aggressive cancer among the group of gynecological malignancies is a leading cause of death in women. OC specific symptoms present at an advanced stage of the disease which leads to late diagnosis. Consequently, prognosis is poor for patients with late presentation of OC symptoms. Standard approach to treatment of OC involves cytoreductive (debulking) surgery, followed by chemotherapy regimen consisting of platinum and taxane based drugs. A major challenge with effective treatment of OC is the development of chemotherapy resistance by patients after initial remission. In most cases (75%) of women diagnosed at an advanced stage of epithelial ovarian cancer (EOC) which is the most common type of OC, a relapse is usually incurable.

[0005] Triple negative breast cancer (TNBC) is known as the most aggressive and lethal form of breast cancer. Due to its lack of expression of estrogen (ER), progesterone (PR), and the human epidermal grow th factor receptor 2 (HER2) receptors, hormone therapy and HER targeting drugs are ineffective, hence chemotherapy is the major treatment option for TNBC patients. Recurrence is a common phenomenon in the treatment of TNBC. Many TNBC patients relapse after initial remission with chemotherapy drugs and radiation therapy due to resistance and metastasis, ultimately resulting in death of some patients.

[0006] In connection w ith both OC and TNBC, platinum therapies and combination therapies including use of platinum have been employed. Nevertheless, these chemotherapeutic strategies are limited by acquired or intrinsic resistance, lack of potency, toxic side effects, and cross-resistance. Meanwhile, platinum therapy has been associated with induction of resistance and toxic side effects.

[0007] The drawbacks of platinum therapy has prompted the generation of new metalbased anticancer agents. Metal complexes with different mechanisms of action in the biological context hold great promise. The mode of action of metal complexes is usually by covalent modification of biomolecules, non-covalent interaction with target proteins, redox activation by biomolecules, and photosensitizer action. This has propelled the broad application of metal-based compounds in the treatment of diseases such as microbial infection, rheumatoid arthritis, diabetes, and cancer. Alternative metals used in recent metalbased drug discovery include ruthenium, rhenium, osmium, and gold. Additionally,developing metal-based anticancer compounds with a known mechanism of action, well differentiated from cisplatin is essential to overcome cisplatin resistance and improve therapeutic performance.

[0008] It has been a century since Otto Warburg reported his observations that cancer cells have high glycolytic activity even in the presence of oxygen (Warburg effect), hence the earlier assumption that mitochondria metabolism is dysfunctional in cancer cells. In 1956, Sidney Weinhouse published one of the earliest criticism of the Warburg effect and several decades dow n the line extensive studies have led to better understanding of the mechanisms of mitochondria function and the role it plays in tumor initiation and progression.

[0009] Beyond its definition as the powerhouse of the cells due to its major role of ATP production via oxidative phosphorylation (OXPHOS), the mitochondria also control several vital cellular processes including regulation of redox homeostasis, regulation of apoptotic cell death, production of reactive oxygen species which are important for cell signaling, and production of metabolites important for cell survival. Structurally, the mitochondria organelle is a tubular netw ork that undergoes coordinated events of fusion and fission. Current understanding is that the balance between the fusion and fission cycles known as mitochondria dynamics control mitochondria metabolism and apoptosis induction. Recent studies have shown that mitochondria dynamics are altered in tumors as a survival strategy. Furthermore, mitochondria structural proteins like the fusion and fission proteins have been implicated in the chemosensitivity or resistance of certain cancers, such as OC.

[0010] Accumulating evidence shows that aggressive tumors have strong dependence on both glycolytic and oxidative pathways. Thus, a promising strategy for developing improved cancer therapy involves targeting mitochondrial metabolism for cancer therapy. For example, it has been shown that combining BACH1 targeting with OXPHOS inhibition by metformin is an effective TNBC therapy. The lack of potency of metformin has prompted several groups to develop superior inhibitors of OXPHOS as therapeutic agents for several diseases including cancer. A small-molecule, Gboxin, has been demonstrated for use as an inhibitor of OXPHOS to target glioblastoma. This work revealed that aggressive tumors rely on mitochondrial respiration for their energy needs, which can be exploited for therapeutic gains. Together these studies illuminate mitochondria as an attractive target for aggressive cancers.

[0011] To target the mitochondria and related mitochondrial processes including, mitochondrial OXPHOS, fission / structure, and nutophagy. rationally designed smallmolecules capable of perturbing mitochondria function selectively are required. Gold(I) N- heterocyclic carbene compounds have been demonstrated for use as mitochondria-targeted anticancer agents in cancer cells. There is potential for the gold compounds to depolarize mitochondria membrane potential in cancer cells. Gold possesses a high redox potential with the ability to react with biological selenols / thiols. Auranofm, an FDA approved drug for rheumatoid arthritis, binds thiols and selenols of proteins including thioredoxin. It has been explored for other medical applications, including clinical trials, as a broad-spectrum antiparasitic or anticancer agent.

[0012] Overall, gold compounds are an attractive new class of anticancer agents, which are well tolerated in mammalian systems and possess a differentiated mechanism of action from that of platinum drugs. Efforts to further utilize gold in medical therapy have been limited by the lack of facile gold-based synthetic strategies and corresponding chemically diverse gold-based compound libraries. Thus, while gold compounds are resurging as an important class of therapeutics across a broad range of pathophysiology, major challenges in gold-based bioinorganic chemistry' remain, including: (i) the kinetic lability' of gold(lll) compounds under physiological conditions; (ii) the lack of a rational design of gold-based drugs; (iii) affinity for thiol-rich proteins and enzymes; (iv) incomplete understanding of gold reagent localization in living systems; and (v) difficulty in accessing stable high-valent gold(m) compounds.

[0013] Significant progress in understanding the structure and reactivity of selected gold(m) complexes has provided new impetus for the use of gold compounds. Previous and ongoing contributions to surmount challenges associated with physiologically relevant gold compounds as anticancer libraries through innovative chemistries continue to expand via the development of new scaffolds including gold-porphyrin, gold-phosphine. and cyclometalated gold variations. Additional synthetic efforts have led to the generation of [CANJ- cyclometalated gold(m) compounds, which present strong sigma-donation of electrons to stabilize the gold center. Furthermore, the repertoire of gold-based agents was expanded with the synthesis of distorted gold constructs bearing chiral phosphine ligands.

[0014] Metastasis is responsible for -90% of cancer related deaths. Aggressive tumor ty pes, including TNBC and OC, often lead to metastasis with limited treatment options. Thismedically unmet need requires innovative drug discovery for improved anticancer therapy. Such cancers adopt increased mitochondrial biogenesis and metabolic activity to promote growth and aggressiveness. Thus, inhibition of mitochondrial activity or uncoupling of mitochondrial respiration is an attractive therapeutic target. However, the clinical efficacy of modulators of mitochondrial dynamics including the electron transport chain and mitochondrial structure is limited by inadequate potency, off-target effects, and poor pharmacokinetics. Potent small-molecules to specifically control mitochondrial function in aggressive tumors holds promise, but none are yet described in the art.

[0015] Iron is an important metal in biology with several cellular processes including respiration. ATP production, and ROS removal. Thus, iron metabolism is crucial in physiology, but its disruption leads to related disorders. The mammalian heme oxygenase family of proteins, HM0X1 (heme monoxygenase 1) and HM0X2 (heme monoxygenase 2) are involved in iron recycling and heme catabolism by catalyzing the stereospecific degradation of heme to biliverdin, carbon monoxide, and free iron. Whereas HM0X1 is inducible and provides a response to oxidative stress, HM0X2 is constitutively expressed and implicated in physiological signaling, cell proliferation, and tumor initiation. Targeting HM0X2 represents a novel strategy7in the treatment of metabolic diseases including cancer.

[0016] Effective cancer therapies in the clinic cave to drug resistance and tumor relapse presenting enormous challenges to patient care. Protein targets in the context of homeostasis and mitochondrial metabolism that can serve as nodes of multiple signaling networks in each disease indication will likely overcome current oncology challenges and promote precision cancer medicine. Therefore, identifying targets with metabolic and homeostatic implications will be ideal and is an unmet need. Recent studies have implicated HM0X2 in the regulation of mitochondria processes and targeting human HM0X2 can treat tumor initiating cells with stem-like character. Studying the therapeutic impact on cellular homeostasis and mitochondria is crucial to attaining clinically relevant therapies.

[0017] Initial discovery of the antifungal ketoconazole as a nonselective inhibitor of HM0X2 accelerated the search for azole-based modulators of HM0X2. Subsequently, the benzimidazole compound, clemizole (l-(4-chlorobenzyl)-2-(pyrrolidin-l-ylmethyl)-lH- benzimidazole) emerged as a selective inhibitor of HM0X2. Other activators of HM0X2 including menadione, and Vitamin KI, K2, K3 have been reported. Recently, a near infra-red probe was identified as targeting HM0X2 with the ability7to treat tumor initiating cells.Despite these discoveries, agents targeting HM0X2 are not in clinical development and the mechanism of small molecule modulation of HM0X2 in the context of a disease is not fully understood. Elucidating the mode of action of HM0X2 targeting in cancer at the molecular level is of pressing need.

[0018] A small population of cells known as cancer stem-like cells (CSCs) have been implicated as major drivers of chemotherapy resistance and tumor recurrence in cancer patients. CSCs, also known as tumor initiating cells, possess capabilities for self-renew al and chemotherapy resistance, hence they can repopulate the original tumor. The failure of the current standard of care chemotherapy drugs to effectively eliminate CSCs along with the bulk tumor cells represents a major clinical challenge in the treatment of cancer patients, hence there is need for more effective chemotherapeutic agents.

[0019] Accordingly, there is a need in the art for compounds, compositions, and methods useful for modulating mitochondrial function, for conferring anti-cancer activity, and / or for treating cancer, including aggressive cancers.SUMMARY

[0020] The presently-disclosed subject matter meets some or all of the above-identified needs, as will become evident to those of ordinary skill in the art after a study of information provided in this document.

[0021] The present application describes a series of gold (III) macrocyclic compounds, compositions comprising such compounds, methods of making such compounds, and methods of using such compounds.

[0022] The presently-disclosed subject matter includes a compound having the following formula:X is selected from the group consistingbond, -NH-alkyl-(C=O)-, -(O)n- where n is 1-8, and -(CH2)n- where n is 1-8; each R is independently selected from the group consisting of: H, -NO2, -NH2, -NR^, -OH, -OR1, - O(C=O)R1, -NH(C=O)R1, -CH3. -(CH2)nCH3 where n is 1-9, Rx, -(C=O)H, -(C=O)R1,a functional moiety.

[0023] As disclosed herein, compounds of the presently-disclosed subject matter exhibit anticancer activity against various aggressive cancer cell types, including triple negative breast cancer (TNBC) and ovarian cancer (OC), for which there is a particular need for improved chemotherapeutic agents. The presently-disclosed compounds also address the need for treatments that target cancer stem cell (CSC) populations, which are often responsible for chemotherapy resistance and tumor recurrence. The presently-disclosed compounds can induce rmtochondnal stress, impacting bioenergetics and leading to decreased ATP -linked respiration and spare respiratory capacity, which are important for cancer cell survival. The compounds result in mitochondrial membrane depolarization, disruption of mitochondrial function, and anti-cancer activity. As also disclosed herein, in vivo studies have shown that the presently-disclosed compounds reduce tumor growthwithout significant changes in body weight, indicating its efficacy and tolerability as a therapeutic agent. Biodistribution analysis suggests a clearance pathway via the kidney and liver, with reduced adverse effects.

[0024] The presently-disclosed compounds include embodiments having a functional moiety. As will be appreciated by those of ordinary skill in the art, depending on the circumstances, the selected functional moiety could be a targeting moiety, a labeling moiety, a therapeutic moiety, a click-chemistry moiety, a purification moiety, or other useful moiety. For example, in some embodiments, the functional moiety could be a fluorophore, such as rhodamine or fluorescein. For another example, the functional moiety could be a peptide, such as denileukin diftitix. leuprolide, thyrotropin alfa, or palifermin. For another example, the functional moiety could be a drug, which could be an anti-cancer drug such as taxol. For another example, the functional moiety could be an antibody, which could be an antibody with anti-cancer utility, such as trastuzunab, pembrolizumab, or cetuximab. For another example, the functional moiety could be an alkyne handle. In one embodiment, for example, the functional moiety is biotin, creating a biotinylated probe of the gold (III) compound, which can be used to facilitate the identification of molecular targets, advancing the understanding of the mechanism of action. In this regard, chemoproteomic profiling with the biotinylated probe has identified HM0X2 as a highly ranked differentially expressed protein, providing insights into the protein pathway interactome associated with HM0X2.

[0025] Accordingly, the presently-disclosed subj ect matter overcome a number of existing challenges in the art by providing compounds, compositions, and methods for targeting mitochondrial dysfunction, identifying molecular targets, inhibiting CSCs, and showing promising in vivo efficacy and safety profiles.

[0026] This Summary describes several embodiments of the presently-disclosed subject matter, and in many cases lists variations and permutations of these embodiments. This Summary is merely exemplary of the numerous and varied embodiments. Mention of one or more representative features of a given embodiment is likewise exemplary. Such an embodiment can typically exist with or without the feature(s) mentioned; likewise, those features can be applied to other embodiments of the presently-disclosed subject matter, whether listed in this Summary or not. To avoid excessive repetition, this Summary does not list or suggest all possible combinations of such features.BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention \w 11 be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are used, and the accompanying drawings of which:

[0028] FIG. 1A-1F. Synthetic scheme for Au-Mac (FIG. 1A); Oak Ridge Thermal Ellipsoid Plot (ORTEP) representation of Au-Macl rendered using POV-Ray, thermal ellipsoids are at the 50% probability level (FIG. IB); HPLC plots of the complexes Au- Macl, 2 and 3 in RPMI for 24 h (FIG. 1C) (mass spectra, FIG. 2A); Reaction monitoring of Complex Au-Macl reactivity7with GSH (complex 1: GSH = 1: 1) for 24 h time using ESI - LC / MS (FIG. ID) (mass spectra, FIG. 3A); Stability of Au-Macl in mouse serum: HPLC chromatogram over 24 h (FIG. ID); and Stability of Au-Macl in mouse serum: percentage of complex (%) over 24 h. (n=2) (FIG. IF).

[0029] FIG. 2A-2B. Solution chemistry; HPLC chromatogram and MS-ES+- API mass spec of Au-Macl, Au-Mac2 and Au-Mac3 in RPMI over the course of 24 h (FIG. 2A); and Solution chemistry; HPLC chromatogram and MS-ES+- API mass spec of Au-Macl, Au- Mac2 and Au-Mac3 in DMEM over the course of 24 h (FIG. 2B).

[0030] FIG. 3A-3B. HPLC chromatogram and intact mass of Au-Macl [m / z 1104.25] reactivity with GSH in equimolar concentration over 24 h. At 24 h time point, formation of <5% Au(I) bisphosphine [m / z 887.20] and concomitant S-arylated aryl-pyridine motif [m / z 489. 14] (FIG. 3A); and HPLC chromatogram and intact mass of Au-Macl [m / z 1104.25] reactivity with GSH (Au-Macl : GSH=1 : 10) over 24 h. At 24 h time point, formation of Au(I) bisphosphine [m / z 887.20] and concomitant S-arylated aryl-pyridine motif [m / z 489.14] (FIG. 3B)

[0031] FIG. 4A-4B HPLC chromatogram and intact mass of Au-Mac2 [m / z 1093.37] reactivity with GSH in equimolar cncentrtion over 24 h. Au-Mac2 remains over 24 h without formation of adducts (FIG. 4A); and HPLC chromatogram and intact mass of Au-Mac2 [m / z 1093.37] reactivity with GSH (Au-Macl : GSH=1 : 10) over 24 h. At 24 h time point, formation of <5% Au(I) bisphosphine [m / z 887.20] (FIG. 4B).

[0032] FIG. 5A-5B. HPLC chromatogram and intact mass of Au-Mac3 [m / z 1090.27] reactivity with GSH in equimolar cncentrtion over 24 h. Au-Mac3 remains over 24 h without formation of adducts (FIG. 5A); and HPLC chromatogram and intact mass of Au-Mac3 [m / z 1090.27] reactivity with GSH (Au-Macl : GSH=1 : 10) over 24 h. At 24 h time point, formation of Au(I) bisphosphine [m / z 887.20] and concomitant S-arylated aryl-pyridine motif [m / z 475.2] (FIG. 5B).

[0033] FIG. 6A-6B. Cell viability assay plots for complexes Au-Macl, 2, 3, 4, 5, and ligand in different TNBC cell lines (FIG. 6A); and Cell viability assay plots Au-Macl in ovarian cancer cells (FIG. 6B).

[0034] FIG. 7. Bar chart explains the summary of IC50 values of Au-Macl, 2, 3, 4 and 5 in different TNBCs.

[0035] FIG. 8A-8B. Bar graph featuring the change in Gl, G2 / M, and S phase over 6, 12, and 24 h in MDA-MB-231 cell lines treated with Au-Macl, data are plotted as the mean ± s.e.m. (n = 3) (FIG. 8A); and Bar graph featuring the change in Gl, G2 / M, and S phase over 6, 12, and 24 h in MDA-MB-468 cell lines treated with Au-Macl, data are plotted as the mean ± s.e.m. (n = 3) (FIG. 8B).

[0036] FIG. 9A-9E. Histogram representing the different phases of the cell cycle (Gl, G2 / M, and S) control and treated with vehicle (DMSO) in MDA-MB-231 (FIG. 9A); Histogram representing the different phases of the cell cycle (Gl, G2 / M, and S) treated with 0.5 pM and 1 pM of Au-Macl (MDA-MB-231) (FIG. 9B); Histogram representing the different phases of the cell cycle (Gl, G2 / M, and S) control and treated with vehicle (DMSO) ( MDA-MB-468) (FIG. 9C); Histogram representing the different phases of the cell cycle (Gl, G2 / M, and S) treated with 0.5 pM of Au-Macl in MDA-MB-468 (FIG. 9D); and Histogram representing the different phases of the cell cycle (Gl. G2 / M, and S) treated with 1.0 pM of Au-Macl in MDA-MB-468 (FIG. 9E).

[0037] FIG. 10A-10B. Bar graph illustrating early-stage apoptotic cell population of Au- Macl in MDA-MB-231 (FIG. 10A) and MDA-MB-468 (FIG. 10B), data are plotted as the mean ±s.e.m. (n = 3).

[0038] FIG. 11A-11B. Apoptosis analysis plot for Au-Macl in MDA-MB-468 (FIG. 11 A) and MDA-MB-231 (FIG. 11B).

[0039] FIG. 12A-12B. Flow cytometry7of tetramethylrhodamine, ethyl ester (TMRE) mitochondrial membrane potential assay analyzing Au-Macl in MDA-MB-468 cells (FIG. 12A); and Bar chart of the effect of Au-Macl on MMP extrapolated from TMRE dye assay using flow cytometry7(FIG. 12B). Data were analyzed by' two-way ANOVA (****p < 0.0001.)

[0040] FIG. 13A-13D. Protein target profiling of Au-Macl. Workflow of target protein identification using biotinylated Au-Macl followed by streptavidin capture and LC-MS / MS (FIG. 13A). Volcanic plot displaying differentially expressed proteins (FIG. 13B). STRING analysis of protein association network related to HM0X2 (FIG. 13C). Immunoblotting of pull-down validation of HM0X2 as the target of Au-Macl -biotin (FIG. 13D). The input blot is indicative of the amount of HM0X2 in the lysates.

[0041] FIG. 14A-14D. Cellular thermal-shift assay (CETSA). Western blot image of CETSA of HM0X2 at the indicated temperatures (FIG. 14A). Stabilization of HM0X2 after incubating cells with Au-Macl (FIG. 14B). CETSA curve showing Au-Macl stabilization of HM0X2 (FIG. 14C). Concentration dependent stabilization of HM0X2 by Au-Macl (FIG.14D)

[0042] FIG. 15A-15F. Immunoblotting to monitor the expression of fusion protein OPA1 (FIG. 15A); Immunoblotting to monitor the expression of mitofusin protein MFN 1 (FIG. 15B); Immunoblotting to monitor the expression of mitophagy marker (FIG. 15C); Immunoblotting to monitor the expression of autophagy markers (FIG. 15D); Flow cytometry analyses of the apoptotic effect of Au-Macl (MDA-MB-468 data is shown (FIG. 15E). Data plotted as the mean ± S.D (n=3) ****. p < 0.0001; ***, p < 0.001; **, p < 0.01; *, p < 0.05 F) Cell titer gio analyses of the effect of cell death suppressors Ferrostatin, Necrostatin and zVAD ( Data plotted as the mean ± S.D (n=3)).

[0043] FIG. 16A-16C. Seahorse bioenergetic stress test of Au-Macl in MDA-MB-468, Normalization of OCR was done per 1,000 cells. Data are plotted as the mean ± SEM, n = 4 (FIG. 16A); Normalized ECAR data extrapolated from Seahorse metabolism analysis (FIG. 16A); Bar graphs showing Basal OCR (FIG. 16C), maximum OCR (FIG. 16D), and ATP- linked OCR (FIG. 16E) readings extrapolated from Seahorse metabolism analysis, Data are plotted as the mean ± SEM (n = 4). Data were analyzed by two-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n.s. = not significant).

[0044] FIG. 17. Plot for mitochondria stress test performed with pneumatic injection of Au-Macl. Bar graphs illustrating the key bioenergetic parameters. All the data were plotted as ± s.e.m. (n = 3).

[0045] FIG. 18A-18E. Schematic representation of the in vivo efficacy study of Au- Macl in BALB / c mouse model (FIG. 18A): In vivo tumor growth inhibition measured by tumor volume (FIG. 18B): Tolerability of Au-Macl measured by body weight (Data plotted as mean ± SEM , n = 4 mouse per group. Two-sided Student’s t test, p values are relative to vehicle control; * 0.01 < p < 0.05 (FIG. 18C)); Biodistribution of complex Au-Macl, Quantification of gold concentration in organ tissue measured using GF-AAS (n=2) (FIG. 18D); Hematoxylin and eosin staining of different organ tissues of Au-Macl treated and untreated mouse (FIG. 18E).

[0046] FIG. 19. Standard curve for biodistribution analysis using GF-AAS.

[0047] FIG. 20A-20F. Au-Macl inhibits TNBC and TNBC-CSC proliferation. Cell viability of TNBC cells treated with Au-Macl over the course of 72 h (FIG. 20A). Effect of Au-Macl on spheroid formation in MDA-MB-468 and SUM 159 cells (FIG. 20B- 20C). Validation of sorted CD447CD24' stem cell population from SUM159 cells (FIG. 20D). Cell viability of sorted CD44+ / CD24'stem cell population from SUMI 59 cells treated with Au-Macl over the course of 72 h (FIG. 20E). Effect of Au-Macl on spheroid formation in sorted SUM 159 CD44VCD24’ stem cell (FIG. 20F).

[0048] FIG. 21A-21F. Au-Macl inhibits mitochondrial respiration in TNBC and TNBC- CSC. Mito stress test of Au-Macl in SUMI 59 cells (FIG. 21A). Extrapolated data from Mito stress test of SUM159 cells treated with Au-Macl. Data are plotted as the mean ± s.e.m. (n =6). Data were analyzed by ordinary one-way ANOVA followed by Dunnett’s multiple comparison test ( **** p < 0.0001. n.s. = not significant) (FIG. 2 IB). Mito stress test of Au- Macl in isolated SUM 159 stem cells (FIG. 21C). Extrapolated data from Mito stress test of isolated SUM159 stem cells treated with Au-Macl. Data are plotted as the mean ± s.e.m. (n =6). Data were analyzed by ordinary one-way ANOVA followed by Dunnett’s multiple comparison test ( **** p < 0.0001. n.s. = not significant) (FIG. 2 ID). Western Blot showing the effect of Au-Macl on the expression of PGC-la , C-myc and p62 in SUM159 and MDA- MB-468 cells (FIG. 21E, 21F).

[0049] FIG. 22A-22B. Regulatory effect of HM0X2 on p62 and MYC. Knockdown of HM0X2 and its effect on p62 and MYC in MDA-MB-468 cells (FIG. 22A) and SUM159 cells (FIG. 22B) examined by Western blot.

[0050] FIG. 23A-23C. Intraperitoneal treatment of Au-Macl (LR13) promote survival in a SUM159 triple negative breast cancer xenograft model. Relative tumor volume is reduced in mice with established SUM159 xenografts treated with LR13 (5 mg / kg) three times per week (Au-Macl, squares) (n = 5 per group) (FIG. 23A). Body weight of mice treated with vehicle or Au-Macl during the study (FIG. 23B). Images of tumor bearing mice treated with vehicle control or Au-Macl (arrow7indicates tumor) (FIG. 23C).

[0051] FIG. 24A-24D. Au-Macl inhibits OC proliferation. Cell viability of OC cells treated with Au-Macl over the course of 72 h. Mean ± s.e.m. n = 3 (FIG. 24A). Effect of Au- Macl on colony formation of UWB1.289 and UWB1.289-PlatR cells (FIG. 24B). Effect of Au-Macl on spheroid formation in UWB 1.289 cells (FIG. 24C). Effect of Au-Macl on spheroid formation in UWB1.289-PlatR cells (FIG. 24D).

[0052] FIG. 25A-25E. Mitochondrial fusion in OC is inhibited by Au-Macl. Effect of Au-Macl on mitochondria membrane potential in OC examined by TMRE dye fluorescence intensity. Data are plotted as the mean ± SD. (n =3). Data were analyzed by ordinary oneway ANOVA followed by Dunnett’s multiple comparison test (** p < 0.01, *** p < 0.001) (FIG. 25A). mtROS production in Au-Macl treated OC cells measured by MitoSOX (PE channel). Data are plotted as the mean ± SD. (n =3). Data were analyzed by ordinary oneway ANOVA followed by Dunnett’s multiple comparison test(* p < 0.05, ** p < 0.01) (FIG. 25B). Au-Macl accumulation in the mitochondria of UWB 1.289 cells visualized by confocal microscopy. UWB1.289 cells were treated with Au-Macl -Alky ne at 20 pM for Ih. Panels: i) Hoechst as the nuclear counterstain, ii) Au-Macl -Alkyne- Alexa Fluor 488 azide adduct, iii) MitoTracker Red CM-H2XR0S (MTR) alone, iv) Merge of all three channels, v) Merge of Hoechst and Au-Macl -Alkyne, vi) Merge of Au-Macl -Alkyne and MTR (FIG. 25C). Western Blot showing the effect of Au-Macl on mitochondria fusion in OC cells (FIG. 25D). TEM showing the effect of Au-Macl on mitochondria structure in UWB1.289 cells. UWB 1.289 cells were treated at 10 pM for lh:30 mins (FIG. 25E).

[0053] FIG. 26A-26G. Au-Macl inhibits oxygen consumption in OC. Au-Macl inhibits ATP production in UWB1.289 (FIG. 26A) and UWB1.289-PlatR (FIG. 26B) cells. Data areplotted as the mean ± s.e.m. (n =6). Data were analyzed by ordinary one-way ANOVA followed by Dunnett’s multiple comparison test (**** p < 0.0001. n.s. = not significant). Au- Mac 1 inhibits ATP production in OV90 (FIG. 26C) and OVCAR3 (FIG. 26D) cells. Data are plotted as the mean ± s.e.m. (n =6). Data were analyzed by ordinary one-way ANOVA followed by Dunnett’s multiple comparison test (**** p < 0.0001. n.s. = not significant). Effect of Au-Macl on mitochondria respiration in UWB1.289 cells (FIG. 26E. 26F). Data are plotted as the mean ± s.e.m. (n =6). Data were analyzed by ordinary one-way ANOVA followed by Dunnett’s multiple comparison test ( **** p < 0.0001). Western Blot showing the effect of Au-Macl on the expression of C-myc and ATF4 in UWB 1.289 and UWB 1.289- PlatR cells (FIG. 26G).

[0054] FIG. 27A-27F. MFN1 degradation by Au-Macl is mediated by PINK1 and PARKIN. Western Blot showing the effect of Au-Macl on the expression of C-myc and ATF4 in UWB1.289 and UWB1.289-PlatR cells (FIG. 27A). Effect of Au-Macl on the expression of OPA1 and MFN1 in UWB1.289 and UWB1.289-PlatR cells pre-treated with MG-132 (FIG. 27B). MFN1 was immunoprecipitated from UWB1.289 and UWB1.289- PlatR cells and subjected to western blot with polyubiquitin antibody (FIG. 27C, 27D). Lysates from Au-Macl treated UWB1.289 and UWB1.289-PlatR cells subjected to immunoblotting to examine phospho-Ser65 ubiquitin (FIG. 27E). Model for Au-Macl induced mitophagy in ovarian cancer (FIG. 27F).

[0055] FIG. 28A-28B. In vivo anticancer activity of Au-Macl. Au-Macl inhibits tumor growth in SKOV-3 xenograft tumor model (FIG. 27A). Mice body weight of Au-Macl treated mice (FIG. 27B).DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0056] The details of one or more embodiments of the presently-disclosed subject matter are set forth in this document. Modifications to embodiments described in this document, and other embodiments, will be evident to those of ordinary skill in the art after a study of the information provided in this document. The information provided in this document, and particularly the specific details of the described exemplary embodiments, is provided primarily for clearness of understanding and no unnecessary limitations are to be understood therefrom. In case of conflict, the specification of this document, including definitions, will control.

[0057] The presently-disclosed subject matter includes a series of gold (III) macrocyclic compounds, compositions comprising such compounds, methods of making such compounds, and methods of using such compounds.

[0058] In some embodiments of the presently-disclosed subject matter, the compound has the following formula:W is selected from the group consisting of , an amino acid, a polypeptide, a nucleotide, and a polyethylene glycol (PEG). X is selected from the group consisting ofselected from the group consisting of covalent bond, -NH-alkyl-(C=O)-, -(O)n- where n is 1 - 8, and -(CH2)n- where n is 1-8. Each R is independently selected from the group consisting of: H, -NO2, -NH2, -NR'2, -OH, -OR1, -O(C=O)R1, -NH(C=O)R1, -CH3, -(CH2)nCH3where nCH3where n=l-8. R2is O or a functional moiety. Q is an anion. In some embodiments, Q is a pharmaceutically-acceptable anion.

[0059] In some embodiments of the presently-disclosed subject matter, the compound has the following formula:polypeptide, a nucleotide, and a polyethylene glycol (PEG). X is selected from the group consistingselected from the group consisting of covalent bond, -NH-alkyl-(C=O)-, -(O)n- where n is 1-8, and -(CFtyln- where n is 1-8. Each R is independently selected from the group consisting of: H, -NO2, -NH2, -NRJ2, -OH, -OR1, -O(C=O)R1, -NH(C=O)R1, -CH3, -(CH2)nCH3where nCH3where n=l-8. R2is O or a functional moiety. Q is an anion. In some embodiments, Q is a pharmaceutically-acceptable anion.

[0060] As disclosed in this application, in some embodiments, a functional moiety can be provided at R2. As will be appreciated by those of ordinary skill in the art, depending on the circumstances, the selected functional moiety could be a targeting moiety, a labeling moiety, a therapeutic moiety, a click-chemistry moiety', a purification moiety, or other useful moiety. For example, in some embodiments, the functional moiety could be biotin. For another example, the functional moiety could be a fluorophore, such as rhodamine or fluorescein. For another example, the functional moiety could be a peptide, such as denileukin diftitix, leuprolide. thyrotropin alfa, or palifermin. For another example, the functional moiety could be a drug, which could be an anti-cancer drug such as taxol. For another example, the functional moiety could be an antibody, which could be an antibody with anti-cancer utility, such as trastuzunab, pembrolizumab, or cetuximab. For another example, the functional moiety could be an alkyne handle. Of course, the forgoing is a limited list of examples offunctional moieties that could be employed, and others will be apparent to one of ordinary skill in the art upon study of this application.

[0061] In some embodiments, the compound has the following formula:

[0062] In some embodiments, the compound has a formula selected from the following:

[0063] In some embodiments, the compound has the following formula:

[0064] In some embodiments, the compound has a formula selected from the following:

[0065] In some embodiments, the compound has the following formula:

[0066] In some embodiments, the compound has the following formula:

[0067] In some embodiments, the compound has the following formula:

[0068] In some embodiments, the compound has the following formula:

[0069] In some embodiments, the compound has the following formula:

[0070] In some embodiments, the compound has the following formula:

[0071] In some embodiments, the compound has a formula selected from the following:

[0072] In some embodiments, the compound has the following formula:

[0073] In some embodiments, the compound has the following formula:

[0074] In some embodiments, the compound of formula (I) or (II), W is an amino acid, a polypeptide, a nucleotide, or a polyethylene glycol (PEG).

[0075] As used herein, the term “amino acid” refers to all canonical amino acids and non- canonical amino acids, as will be recognized by those of ordinary skill in the art. Accordingly, the term is inclusive of alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamine (Gin, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (He, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W). tyrosine (Tyr, Y), valine (Vai, V), selenocysteine (Sec, U), pyrrolysine (Pyl, O), ornithine, citrulline, hydroxyproline, hydroxylysine, gammacarboxyglutamate, N-methyllysine, N-methylarginine, and other noncanonical amino acids. As used herein, “amino acid residue” refers to an amino acid formed upon chemical digestion (hydrolysis) of a polypeptide at its peptide linkages. The amino acid residues described herein are generally in the “L” isomeric form. Residues in the “D” isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property is retained by the polypeptide.

[0076] As used herein, the term “polypeptide” means any polymer comprising any of the 20 protein amino acids, regardless of its size. Although “protein” is often used in reference to relatively large polypeptides, and “peptide” is often used in reference to small polypeptides, usage of these terms in the art overlaps and varies. The term “polypeptide” as used herein refers to peptides, polypeptides and proteins, unless otherwise noted. As used herein, the terms “protein”, “polypeptide,” and “peptide” are used interchangeably. In some cases, the polypeptide includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32. 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids. As will be appreciated by one of ordinary’ skill in the art upon study of this document, in some cases, the polypeptide can be selected due to a desired function. For example, in some cases, the polypeptide has drug targeting functionality.

[0077] As used herein, the terms “nucleotide”, “polynucleotide”, “nucleic acid” and “nucleic acid sequence” refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single or double stranded form. Unless specifically limited, the term encompasses nucleic acids containing know n analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. In some cases, the nucleotide includes 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49. 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74. 75. 76. 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 residues.

[0078] As used herein, the terms “polyethylene glycol” or “PEG” refer to polymers of ethylene oxide (-CH2CH2O-). As will be appreciated by the skilled artisan, the length of the polymer chain can vary depending on the desired properties. In some cases, low molecular weight PEGs can be employed, which are about 200 to 1,000 Da. Low molecular weight PEGs can be used, for example, for solubility enhancement and in liquid preparations. In some cases, medium molecular weight PEGs can be employed, which are about 2,000 to 10,000 Da. Medium molecular weight PEGs can be used, for example, in various formulations including tablets and ointments. In some cases, high molecular weight PEGs can be employed, which are about 20,000 to 40,000 Da. High molecular weight PEGs can be used, for example, for delivery where prolonged systemic circulation is desirable.

[0079] While compounds of formula (I) or (II), in which W is an amino acid, a polypeptide, a nucleotide, or a polyethylene glycol (PEG) will be understood by one of ordinary7skill in the art upon study of this document, the follow are provided for further illustration. For example, in some embodiments, W can be the amino acid lysine, as in the following formula:The compound of the following formula provides another example of an embodiment in which W can be the amino acid lysine:(XXXIV)The compound of the following formula provides another example of an embodiment in which W can be a polypeptide:The foregoing formula W includes lysine and alanine, joined with a peptide bond. As will be apparent to one of ordinary skill in the art. additional or alternate amino acids can be provided, being joined to the compound with further peptide bonds.

[0080] The presently-disclosed subject matter further includes a pharmaceutical composition including a compound as disclosed herein and a pharmaceutically-acceptable carrier.

[0081] The presently-disclosed subject matter further includes methods that make use of a compound or a composition as disclosed herein.

[0082] Some embodiments of the presently-disclosed subject matter include a method of conferring anti-cancer activity to a cancer cell, which involves contacting a cancer cell with an effective amount of a compound or composition as disclosed herein. In some embodiments, conferring anti-cancer activity results in one or more of inhibiting proliferation of the cancer cell, inhibiting metastasis, and killing the cancer cell. In some embodiments, the cancer cell is a cultured cell. In some embodiments, the cancer cell is in a subject. In some embodiments, the cancer is a breast cancer. In some embodiments, the cancer is triple negative breast cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is glioblastoma. In some embodiments, the cancer is platinum resistant cancer. In some embodiments, the cancer is a platinum resistant gynecologic cancer. In some embodiments, the cancer is a platinum resistant ovarian tumor.

[0083] Some embodiments of the presently-disclosed subject matter include a method of modulating mitochondrial function in a cell, which involves contacting a cell with an effective amount of with an effective amount of a compound or composition as disclosed herein. In some embodiments, the cell is a cultured cell. In some embodiments, the cell is in a subject. In some embodiments, the cell is a cancer cell.

[0084] Some embodiments of the presently-disclosed subject matter include a method of inhibiting oxidative phosphorylation (OXPHOS) in a cell, which involves contacting a cell with an effective amount of a compound or composition as disclosed herein. In some embodiments, the cell is a cultured cell. In some embodiments, the cell is in a subject. In some embodiments, the cell is a cancer cell.

[0085] Some embodiments of the presently-disclosed subject matter include a method of reducing viability- of cancer stem cells (CSCs), which involves contacting a cell w ith an effective amount of a compound or composition as disclosed herein. In some embodiments, the cell is a cultured cell. In some embodiments, the cell is in a subject. In some embodiments, the cell is a cancer cell.

[0086] Some embodiments of the presently-disclosed subject matter include a method of targeting HM0X2 in a cell, comprising: contacting a cell with an effective amount of a compound or composition as disclosed herein. In some embodiments, the cell is a culturedcell. In some embodiments, the cell is in a subject. In some embodiments, the cell is a cancer cell.

[0087] While the terms used herein are believed to be well understood by those of ordinary7skill in the art, certain definitions are set forth to facilitate explanation of the presently-disclosed subject matter.

[0088] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the invention(s) belong.

[0089] All patents, patent applications, published applications and publications, GenBank sequences, databases, websites and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety'.

[0090] Where reference is made to a URL or other such identifier or address, it understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability’ and public dissemination of such information.

[0091] As used herein, the abbreviations for any protective groups, amino acids and other compounds, are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (see, Biochem. (1972) 1 1(9): 1726-1732).

[0092] Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the presently-disclosed subject matter, representative methods, devices, and materials are described herein.

[0093] As used herein the term ‘‘alky l'’ refers to Cl-20 inclusive, linear (i.e., “straightchain”), branched, or cyclic, saturated or at least partially and in some cases fully unsaturated (i.e., alkenyl and alkynyl) hydrocarbon chains, including for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, penty l, hexyl, octyl, ethenyl, propenyl, butenyl, pentenyl, hexenyl, octenyl, butadienyl, propynyl, methylpropynyl, butynyl, pentynyl, hexynyl, heptynyl, and allenyl groups. “Branched” refers to an alky l group in which a lower alkyl group, such as methyl, ethyl or propyl, is attached to a linear alkyl chain. “Loweralkyl’’ refers to an alkyl group having 1 to about 8 carbon atoms (i.e., a Cl-8 alky l), 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, in particular, to Cl-8 straight-chain alkyds. In other embodiments, “alkyl” refers, in particular, to Cl-8 branched-chain alkyls.

[0094] Alkyl groups can optionally be substituted (a “substituted alkyd”) with one or more alkyl group substituents, which can be the same or different. The term “alkyl group substituent” includes but is not limited to alkyd, substituted alkyd, halo, arylamino, acyl, hydroxyl, aryloxyl, alkoxyl, alkydthio, arylthio, aralkyloxyl, aralkydthio, carboxyl, alkoxycarbonyl, oxo, and cycloalkyl. There can be optionally inserted along the alkyd chain one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms, wherein the nitrogen substituent is hydrogen, lower alkyd (also referred to herein as “alkydaminoalkyl”), or ary l. Thus, as used herein, the term “substituted alkyd” includes alkyd groups, as defined herein, in which one or more atoms or functional groups of the alkyl group are replaced with another atom or functional group, including for example, alkyl, substituted alkyd, halogen, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, and mercapto. Further, as used herein, the terms alkyd and / or “substituted alkyd” include an “allyl” or an “allylic group.” The terms “allylic group” or “allyl” refer to the group CH2HC=CH2 and derivatives thereof formed by substitution. Thus, the terms alkyl and / or substituted alkyl include allyl groups, such as but not limited to, allyl, methylallyl, dimethylallyl, and the like. The term “allylic position” or “allylic site” refers to the saturated carbon atom of an allylic group. Thus, a group, such as a hydroxyl group or other substituent group, attached at an allylic site can be referred to as “allylic.”

[0095] The term ‘ary l’ is used herein to refer to an aromatic substituent that can be a single aromatic ring, or multiple aromatic rings that are fused together, linked covalently, or linked to a common group, such as, but not limited to, a methylene or ethylene moiety. The common linking group also can be a carbonyl, as in benzophenone, or oxygen, as in diphenyl ether, or nitrogen, as in diphenylamine. The term “aryl” specifically7encompasses heterocyclic aromatic compounds. The aromatic ring(s) can comprise phenyl, naphthyl, biphenyl, diphenylether, diphenylamine and benzophenone, among others. In particular embodiments, the term “aryl” means a cyclic aromatic comprising about 5 to about 10 carbonatoms, e.g.. 5, 6, 7, 8, 9, or 10 carbon atoms, and including 5- and 6-membered hydrocarbon and heterocyclic aromatic rings.

[0096] The aryl group can be optionally substituted (a “substituted aryl”) with one or more aryl group substituents, which can be the same or different, wherein “aryl group substituent” includes alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, hydroxyl, alkoxyl, aryloxyl. aralkyloxyl, carboxyl, acyl, halo, nitro, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acyloxyl, acylamino, aroylamino, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, arylthio, alkylthio, alky lene, and -NR’R‘, wherein R’ and R’ can each be independently hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, and aralkyl. Thus, as used herein, the term “substituted aryl” includes aryl groups, as defined herein, in which one or more atoms or functional groups of the aryl group are replaced with another atom or functional group, including for example, alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, and mercapto.

[0097] Specific examples of aryl groups include, but are not limited to, cyclopentadienyl, phenyl, furan, thiophene, pyrrole, pyran, pyridine, imidazole, benzimidazole, benzothiazole, isothiazole, isoxazole, pyrazole, pyrazine, triazine, pyrimidine, quinoline, isoquinoline, indole, carbazole, and the like.

[0098] As used herein, an “anion” or “counter anion” refers to the negatively charged ion that pairs with a positively charged ion (cation) of the presently-disclosed compounds. As will be appreciated by one of ordinary skill in the art upon study of this document, the anion can be a “pharmaceutically acceptable anion.” Examples of such anions include chloride, citrates, tartrates, acetates, sulfates, phosphates, benzoates, maleates, fumarates, gluconates, and mesylates. These anions can be selected based on their ability to enhance the solubility, stability', or bioavailability of active pharmaceutical ingredients, each contributing distinct properties beneficial for various drug formulations. For example, chloride (Cl ) is highly soluble and generally very stable, making it suitable for a wide range of pharmaceutical applications, including injectable and oral formulations; citrates (CeHsO?3) enhance solubility7, have a buffering capacity' to maintain the pH of formulations, and can be used for their metal chelating properties; tartrates (CfFEOe2) are useful for their good solubility and chelating properties, which can improve the stability and taste of oral medications; acetates (CH3COO ) can be used for their solubility, buffering capacity, and can also help stabilize volatile or reactive active pharmaceutical ingredients; sulfates (SOf ) can be used due totheir solubility and non-toxic nature, and are included in many injectable and oral formulations; phosphates (PCh3) can be used for their buffering capacity and stability, and they are beneficial in formulations sensitive to pH fluctuations; benzoates (CeHsCOO ) can be used for their antimicrobial properties, which can extend the shelf life of pharmaceutical products, in addition to enhancing solubility; maleates (C4H3O42) can be used to improve solubility and stability, particularly in oral formulations, and sometimes for their tastemodifying properties; fumarates (C4H2O42) can be used to enhance solubility and bioavailability; gluconates (CsHnO? ) can be used for their chelating abilities; and mesylates (CH3SO3 ) can be used to improve solubility7and absorption in the gastrointestinal tract.

[0099] The present application can '’comprise" (open ended) or “consist essentially of’ the components of the present invention as well as other ingredients or elements described herein. As used herein, “comprising” is open ended and means the elements recited, or their equivalent in structure or function, plus any other element or elements which are not recited. The terms “having” and “including” are also to be construed as open ended unless the context suggests otherwise.

[0100] Following long-standing patent law convention, the terms “a”, “an”, and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to “a cell” includes a plurality of such cells, and so forth.

[0101] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently-disclosed subject matter.

[0102] As used herein, the term “about,” when referring to a value or to an amount of mass, weight, time, volume, concentration or percentage is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%. in some embodiments ±0. 1%, in some embodiments ±0.01%, and in some embodiments ±0.001% from the specified amount, as such variations are appropriate to perform the disclosed method.

[0103] As used herein, ranges can be expressed as from “about"’ one particular value, and / or to “about"’ another particular value. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0104] As used herein, “optional” or “optionally” means that the subsequently described event or circumstance does or does not occur and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, an optionally variant portion means that the portion is variant or non-variant.

[0105] As used herein, the term “pharmaceutically acceptable carrier’ refers to sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use.

[0106] As used herein, the term “effective amount” refers to an amount that is sufficient to achieve the desired result or to have an effect on an undesired condition.

[0107] As used herein, the term “subject” refers to a target of administration. The subject of the herein disclosed methods can be a human or non-human animal. Thus, veterinary uses are provided in accordance with the presently disclosed subject matter. As such, the presently disclosed subject matter provides for administration to mammals such as humans and non-human primates, as well as those mammals of importance due to being endangered, such as Siberian tigers; of economic importance, such as animals raised on farms for consumption by humans; and / or animals of social importance to humans, such as animals kept as pets or in zoos. Examples of such animals include but are not limited to: carnivores such as cats and dogs; swine, including pigs, hogs, and wild boars; ruminants and / or ungulates such as cattle, oxen, sheep, giraffes, deer, goats, bison, and camels; rabbits, guinea pigs, and rodents. Also provided is the treatment of birds, including the treatment of those kinds of birds that are endangered and / or kept in zoos, as well as fowl, and more particularly domesticated fowl, i.e., poultry, such as turkeys, chickens, ducks, geese, guinea fowl, and the like, as they are also of economic importance to humans. Thus, also provided is the treatment of livestock, including, but not limited to, domesticated swine, ruminants,ungulates, horses (including race horses), poultry, and the like. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered.

[0108] The presently-disclosed subject matter is further illustrated by the following specific but non-limiting examples. The following examples may include compilations of data that are representative of data gathered at various times during the course of development and experimentation related to the present invention.EXAMPLES

[0109] Example 1: Materials.

[0110] All the reagents and solvents used in this work were bought from commercial vendors (Acros, Millipore- Sigma, TCI-America, Alfa Aesar, and Pharmco Aaper USA) and were used without additional purification unless explicitly stated otherwise. HAuCh’SfLO was purchased from NANOP ARTZ and stored in a glovebox or desiccator before use. 2- benzoylpyridine, 2-benzylpyridine, 2-phenylpyridine, 2-phenoxypyridine, and Benzo [h] quinoline ligands w ere purchased from the following commercial vendors Sigma Aldrich, TCI America, and Alfa Aesar. (R,R)-DACH phenyl Trost ligand was purchased from Santa Cruz. Deuterated solvents for NMR were acquired from Cambridge Isotope Laboratories and used as is. Compounds were purified by silica gel (SiliCycle, P / N: R10030B SiliaFlash F60, size: 40-63 pm, Canada) for column chromatography. Analytical thin-layer chromatography (TLC) was performed by aluminum backed silica-gel plates (20 x 20 cm2, TLA-R10011B-323) from SiliCycle. Reactions were conducted under standard atmospheric conditions. Progress of reactions was evaluated by NMR and TLC. Low - wavelength light (254 nm) w as used to visualize the TLC plates.

[0111] Example 2: Physical measurements.

[0112] rH NMR and13C {1H} NMR spectra were collected on a 500 MHz Bruker spectrometer, a 400 MHz Varian spectrometer, or a 500 MHz JEOL ECZr at the University' of Kentucky (UK) NMR Center. Solvent signals were used as internal reference t'H NMR: DMSO at 5 = 2.50 ppm and CDjOD at d = 3.31;13C NMR: DMSO at 5 = 39.52 ppm and CD3OD at 5 = 49.00 ppm) for chemical shifts in 'H NMR spectra collected. X-ray crystal structures w ere obtained from the UK X-ray cry stallography center at the Department ofChemistry. Synergy Hl hybrid plate reader at College of Pharmacy (UK) was used for cell viability experiment. Flash silica-gel chromatography was performed using Combi-Flash from Teledyne ISCO. An Agilent 1100 series HPLC instrument connected to Advion (reverse-phase C18 column) was used to obtain the trace HPLC data and low-resolution ESI - MS spectrum. Flow cytometry' data were obtained from UK flow cytometry' and immune monitoring core facility using CytoFLEXLX. Mitochondrial bioenergetics were studied using UK RM SRF core facility using Seahorse XFe96 analyzer.

[0113] Example 3: Synthetic procedures.

[0114] Synthesis of cyclometalated gold(III) complexes. [(CAN)Au(III)-(pcp) Ch] [pep= 2-(2-pyridylcarbonyl)phenyl], [(CAN)Au(IH)-(byp)Cl 2] [byp=2 -benzylpyridine], (CAN)- phenyl,2-pyridyl ether- Au(III)C12, [(CAN)Au(III)-(PhPy) ChHPhPy = 2-phenylpyridine] and [(CAN)Au(III) -benzo(h) quinoline-Ch] were synthesized using heating mantle equipped with pressure tube using high temperature -270 °C.

[0115] Example 4: Synthesis of Au-Macl.

[0116] To a 25 mL round-bottom flask, [CAN] Au(III)- (pep) CI2] (mg, mmol), (R,R)- DACH phenyl Trost ligand (69.4 mg, 0.2 mmol, 2 equiv). and 10.0 mL of MeOH / DCM (v / v = 1: 1) were added and stirred for 2-4 h at room temperature. A pale-yellow clear solution was formed. The progress of the reaction was monitored by using TLC. The solvent was evaporated in vacuo and the crude was redissolved in minimal amount of DCM and precipitated with diethyl ether, (y ield = 80 %). 'H NMR (400 MHz. CDCh-rf) 5 9. 16 (s, 1H), 9. 10 - 9.06 (m. 1H), 8.54 (d. J= 7.8 Hz, 1H), 8.44 (d, J= 7.7 Hz, 1H), 8.34 (d, J= 4.7 Hz, 1H), 8.28 (d, J= 7.9 Hz, 1H), 7.63 - 7.50 (m, 5H), 7.46 (d, J= 7.6 Hz, 2H), 7.35 (d, J= 7.6Hz, 2H), 7.14 (d, J= 7.2 Hz, 2H), 7.02 (dt, J= 26.5, 8.0 Hz, 5H), 6.92 - 6.81 (m, 4H), 6.76 - 6.61 (m, 7H), 6.36 - 6.29 (m, 1H). 5.28 (d, J= 10.4 Hz. 1H), 3.87 (s, 1H), 1.97 (t, J= 12.7 Hz, 2H), 1.70 (s, 2H), 1.60 (d, J= 12.2 Hz, 1H), 1.48 (d, J= 12.7 Hz, 1H), 1.35 (t, J= 12.7 Hz, 1H).31P pH} NMR (162 MHz, CDCh-d) 5 35.97, 35.68.13C pH} NMR (101 MHz, CDCh-rf) 5 194.36, 165.81, 165.45, 153.92, 147.63, 135.90, 135.29, 132.65, 132.44, 132.26, 131.13. 131.00, 130.04, 129.81, 128.16, 125.64, 124.78, 124.47, 77.33, 77.01, 76.69. 55.89, 50.76, 33.64. 30.94, 25.32.MS - ES+- API : m / z , z = 2 (%) 552.9 (100), calculated / » z. z = 2 for [M+] 552.12 (100). Elemental Anal. % Calcd for C56H48AUCI2N3O3P2.O.O5 CH2CI2: C, 58.79%; H, 4.23%; N, 3.67%. Found: C, 58.51%; H, 4.53%; N, 3.64%.HPLC method. Flow rate: 1 mL / min; X = 260 nm; Eluent A = H2O with 0.1% TFA; Eluent B = ACN with 0.1 % Formic acid; Solvent Gradient: 0 - 15 min (0: 100 H2O: ACN), 5 min (100:0 H2O: ACN).

[0117] Example 5: Synthesis of Au-Mac2.

[0118] Same procedure from synthesis of Au-Macl was followed using (CAN) 2- phenoxypyridine -Au (III)C12. (yield = 78 %).1H NMR (400 MHz, CDCh-t / ) 5 9.32 (d, J= 8.9 Hz. 1H), 8.94 (d. J = 6.4 Hz. 1H), 8.60 - 8.53 (m, 1H), 8.47 - 8.41 (m, 1H), 7.76 (d, J = 8.0 Hz, 1H), 7.67 - 7.51 (m, 4H), 7.43 - 7.34 (m, 2H), 7.35 - 7.27 (m, 5H), 7.14 (t, J= 7.6 Hz, 3H), 7.06 - 6.87 (m, 9H), 6.80 - 6.65 (m, 8H), 6.47 (td, J = 8.2, 4.4 Hz, 1H), 5.30 (td, J = 10.5, 9.9, 3.4 Hz. 1H), 3.93 (p. J= 7.2 Hz. 1H), 3.46 (s, 2H), 1.75 - 1.62 (m, 3H), 1.54 - 1.34 (m. 2H).31P pH} NMR(162 MHz. CDCl3-rf) 5 35.09, 34.87.13C pH} NMR (101 MHz, CDCW) 5 166.09, 165.36, 161.72, 154.34, 146.99, 138.31, 137.83, 136.92, 135.18, 134.84, 134.19, 133.70, 132.45, 132.39, 131.22, 130.17, 129.98, 128.89, 128.09, 127.74, 127.73, 126.56, 119.59, 118.08, 112.23, 77.32, 77.00, 76.69, 55.88, 50.65, 33.47, 30.77, 25.29. MS - ES+- API: m 'z, z = 2 (%) 546.10 (100), calculated m / z, z = 2 for [M+] 546.12 (100).Elemental Anal. % Calcd for C55H48AUCI2N3O3P2.I.O5 CH2CI2: C, 55.27%; H, 4.15%; N, 3.45%. Found: C, 55. 1%; H, 4.35%; N, 3.44%.HPLC method. Flow rate: 1 mL / min; X = 260 nm; Eluent A = H2O with 0.1% TFA; Eluent B = ACN with 0.1% Formic acid; SolventGradient: 0 - 15 mm (0: 100 H2O: ACN), 15-20 mm (100:0 H2O:ACN).

[0119] Example 6: Synthesis of Au-Mac3.

[0120] Same procedure from synthesis of Au-Mac 1 was followed using (CAN) 2- phenylpyridine -Au(III)Cl2. (yield = 76 %). 'H NMR (500 MHz, CDCh-t / ) 5 9.39 (d, J= 8.9 Hz, 1H), 8.94 (d, J= 6.2 Hz, 1H), 8.74 (s, 1H), 8.61 (d, J= 7.9 Hz, 1H), 8.46 (d, J= 7.8 Hz, 1H), 8.29 (s, 1H), 7.92 (d, J= 8.0 Hz, 1H), 7.65 (dt, J = 16.1, 7.5 Hz, 3H), 7.46 (dt, J= 14.7, 7.3 Hz. 4H), 7.40 (t, J= 7.1 Hz, 2H), 7.33 (q, J= 8.2 Hz, 4H), 7.27 (s, 1H). 7.22 (t. J= 7.5 Hz. 2H), 6.97 (ddt. J = 26.7. 17.5. 7.6 Hz. 7H), 6.85 (t, J= 7.6 Hz, 1H), 6.72 - 6.67 (m, 2H). 6.61 (dd, J = 14.9, 7.0 Hz, 5H), 6.42 (d, J= 8.6 Hz, 2H), 5.77 (d, J= 7.7 Hz, 1H), 5.28 - 5.23 (m, 1H), 3.93 (tt, J= 11.4, 5.9 Hz, 1H), 3.37 (d, J= 16.3 Hz, 1H), 3.01 (s, 1H), 1.77 - 1.60 (m, 4H), 1.55 - 1.43 (m, 1H), 1.40 - 1.18 (m, 4H), 0.86 (dt, J= 19.2, 6.3 Hz, 1H).31P pH} NMR (202 MHz, CDCh-rf) 6 33.62. 33.53.13C pH} NMR (101 MHz, CDCh-r / ) 5 166.03. 165.35, 139.13, 138.53, 137.88, 136.46, 136.35, 135.22, 134.87, 134.82, 133.64, 133.23, 132.57, 131.75, 131.12, 130.38, 130.04, 128.95, 128.32, 128.08, 127.48, 126.49, 77.31, 77.00, 76.68, 55.88, 50.67, 33.40, 30.66, 25.24, 25.20. MS - ES+- API: m / z , z = 2 (%) 545.30 (100), calculated m'z, z = 2 for [M+] 545. 13 (100). Elemental Anal. % Calcd for C56H50AUCI2N3O3P2.O.65 C4H10O: C, 59.1%; H, 4.78%; N, 3.53%. Found: C, 59.18%; H, 4.89%; N, 3.64%. HPLC method. Flow rate: 1 mL / min; X = 260 nm; Eluent A = H2O with 0. 1% TFA; Eluent B = ACN with 0.1% Formic acid; Solvent Gradient: 0 - 15 min (0: 100 H2O: ACN), 5 min (100:0 H2O:ACN).

[0121] Example 7: Synthesis of Au-Mac4.

[0122] Same procedure from synthesis of Au-Macl was followed using (CAN) 2- benzylpyridine -Au(III)C12. Silica gel flash column chromatography (eluent 0-5% DCM in MeOH) was used to purify the complex, (yield = 37 %). 'H NMR (400 MHz, CDCU - d) 5 9. 13 (d, J = 6.2 Hz, 1H), 8.93 (d, J = 8.8 Hz, 1H), 8.58 - 8.37 (m, 3H), 7.56 (dq, J = 14.3, 7.4 Hz, 4H), 7.31 (d, J = 7.6 Hz, 1H), 7.25 - 7. 15 (m, 6H), 7.07 (td, J = 7.7, 1.8 Hz, 2H), 7.03 - 6.87 (m, 10H), 6.79 (s. 1H), 6.70 (t, J = 6.3 Hz, 1H), 6.45 (dt. J = 27.0, 8.5 Hz, 3H), 6.09 (ddd, J = 10.4, 7.4, 2.4 Hz, 1H), 3.90 (d, J = 6.3 Hz, 1H), 2.19 (s, 4H), 1.97 (t, J = 13.1 Hz, 2H), 1.84 (td, J = 12.5, 3.5 Hz, 1H), 1.70 (d, J = 22.7 Hz, 2H), 1.64 - 1.55 (m, 1H), 1.55 - 1.42 (m, 1H), 1.42 - 1.25 (m, 2H).31P pH} NMR (162 MHz, CDC13- d) 5 34.73, 33.63.1?C pH} NMR (101 MHz. CDCh-rf) 5 165.68, 165.41, 147.62, 138.19. 137.25, 135.93, 135.00, 133.14. 132.37, 132.11, 131.94, 130.76. 130.54. 130.42, 129.94, 129.89, 129.53, 128.27. 128.20, 128.06, 128.00, 127.19, 127.10, 126.42, 121.68, 77.30, 76.98, 76.67, 55.83, 53.38, 50.89, 33.55, 30.86, 25.39, 25.28. MS - ES+- API: m,z , z = 2 (%) 538.20 (100), calculated in z. z = 2 fox [M+] 538.13 (100).

[0123] Example 8: Synthesis of Au-Mac5.

[0124] Same procedure from synthesis of Au-Mac 1 was followed using (CAN) 2- benzo(h)quinoline -Au(III)C12. Silica gel flash column chromatography (eluent 0-5% DCM in MeOH) was used to purify the complex, (yield = 28 %). 'H NMR (400 MHz, CDCls - d) 3 9.17 (d, .7 = 8,8 Hz. 1H), 8.91 (dd, J= 29.2, 7.8 Hz, 3H), 8.52 - 8.44 (m, 3H), 8.27 - 8.13 (m, 2H), 7.93 (s, 1H), 7.83 - 7.63 (m, 4H), 7.63 - 7.54 (m, 6H). 7.38 (ddd, J= 35.2, 12.3, 5.2 Hz, 10H), 7.19 (dd, J= 15.6, 6.0 Hz, 4H), 7.08 (d, J = 7.3 Hz, 2H), 7.00 (q, J = 7.8 Hz, 2H), 6.93 - 6.81 (m, 2H), 6.77 - 6.54 (m, 8H), 6.40 (t, J = 9.1 Hz, 2H), 6.00 (d, J = 41.7 Hz, 5H), 5.33 - 5.28 (m, 1H), 3.94 (s, 1H), 1.96 (d, J= 13.9 Hz, 4H), 1.71 (d, J = 12.0 Hz, 3H), 1.58 (s, 1H), 1.44 - 1.28 (m, 2H), 1.21 (s, 1H).31P pH} NMR (162 MHz, CDCh - d) 5 31.98, 31.74, 30.20, 30.01.13C pH} NMR (101 MHz. CDCh-tZ) 6 165.76, 165.23, 165.20. 148.60. 137.90, 135.57, 134.99, 134.30, 132.36, 132.31, 131.84, 131.68, 131.09, 130.94, 130.78, 129.86, 129.74, 128.78, 128.72, 128.15, 128.08, 127.41, 126.83, 126.26, 125.61, 124.95, 124.41, 120.80. 120.37, 77.29, 76.97, 76.65, 55.78, 53.37, 51.06, 33.44. 30.60, 25.45, 25.33.

[0125] Example 9: General Procedures for the Synthesis of AuMac Compounds.Scheme 1. Scalable synthesis for AuMac compounds.Scheme 2. Noncanoncal peptide ligand synthesis.Scheme 3. Synthetic scheme for AuMac-biotin. a) (Aminooxy) acetic acid hemihydrochloride, DCM / MeOH, RT, 2 days, b) HOBT, EDC, anh. DMF, RT, overnight, c) (R.R) -DACH phenyl Trost ligand, DCM / MeOH. RT, 2 h.

[0126] Example 10: Synthesis of [CAN]Au(III)-(pcp) Ch] [pep = 2-(2- pyridylcarbonyl)phenyl] (SI).

[0127] To a 25 mL round-botom flask, a solution of HAuC14.3H2O (0.200 g, Immol, 1 equiv) in DI water (10 mL) was added phenyl(pyridin-2-yl)methanone (0.102 g, 1 mmol, 1 equiv). The solution was placed in a pressure tube for 48 hrs. After the 48 hrs, the reaction mixture was filtered under vacuum and the ppt was washed with DI water, Ethanol and Diethyl ether. White solid product was obtained, (yield = 63%).JH NMR (500 MHz, DMSO- / A) 5 9.48 (d. J - 4.7 Hz. 1H), 8.55 (t, J= 7.7 Hz, 1H), 8.37 (d, J= 9.6 Hz, 1H), 8.09 (t, J = 7.7 Hz, 1H), 7.76 (d, J = 7.4 Hz, 1H), 7.69 (dd, J= 6.9, 2.5 Hz, 1H), 7.51 - 7.43 (m, 2H).13C NMR (126 MHz, DMSO-1A) 5 189.24, 153.33, 145.67. 144.88. 137.26, 134.48, 133.76, 130.42, 130.11 , 129.12, 128.32, 127.63.

[0128] Example 11: Synthesis of Au(III) oxime (S2).

[0129] To a 25 mL round-botom flask, a solution of [CAN]Au(III)-(pcp) Ch] (80 mg, 0.2 mmol 1 equiv) in DCM (5 mL) and Methanol (5mL) was added (Aminooxy) acetic acid hemihydrochloride (49 mg, 0.6 mmol, 3 equiv). The solution was placed in a 20 mL vial and stirred for 48 hrs at RT. After 48 hrs, the reaction mixture was filtered under vacuum and the ppt was washed with Methanol 3 times. White solid product was obtained, (yield = 67%).NMR (500 MHz, DMSO-d6) 5 9.31 (d, J = 6.0 Hz, 1H), 8.46 (d, J = 4.3 Hz, 2H), 7.93 (q, J = 5.0 Hz, 1H), 7.51 (d, J = 8.0 Hz, 1H), 7.34 - 7.23 (m, 3H), 4.89 (s, 2H).13C NMR (126 MHz, DMSO-d6) 5 171.11, 153.50, 153.26, 144.24, 143.83, 137.99, 133.87, 130.51, 129.19, 129.04. 128.69, 128.28, 128.12, 72.19.

[0130] Example 12: Synthesis of Oxime-biotin-EDA (S3).

[0131] To a well purged Schlenk flask Biotin - EDA (27.2 mg, 0.095 mmol), oxime (24.8 mg, 0.047 mmol), HOBt (19.34 mg, 0.14 mmol) and EDC (27.46 mg, 0.14 mmol) in 1.0 ml anhydrous DMF was added and stirred overnight at RT. Progress of the recti on was monitored by using TLC. After 24 h, water (10 mL) was added and extracted from DCM / IPA mixture (L I). Organic layers were washed with 30.0 mL of bnne and dried over anhydrous MgSO4. The solvent was evaporated in vacuo. Yield: 25 mg, 33%.1H NMR (400 MHz,DMS0-d6) 5 9.35 (dd, J= 6.0, 1.5 Hz, 1H), 8.70 (dd, J= 8.0, 1.6 Hz. 1H), 8.50 (td, J= 7.8, 1.5 Hz, 1H), 8.24 (t, J= 5.3 Hz, 1H), 8.01 - 7.92 (m, 3H), 7.73 (dt, J = 8.3. 1.0 Hz. 1H), 7.55 (ddd, J= 8.3, 5.4, 1.1 Hz, 2H), 7.44 - 7.41 (m, 1H), 7.38 - 7.34 (m, 2H), 7.34 (s, 1H), 6.43 - 6.35 (m, 2H), 4.77 (s, 2H), 4.29 (dd, J = 7.7, 5.0 Hz, 1H), 4.13 - 4.07 (m, 1H), 3.21 (dt, J = 14.2, 5.6 Hz, 4H), 3.09 - 3.02 (m, 1H), 2.80 (dt, J= 12.4, 5.3 Hz, 1H), 2.56 (d, J= 12.4 Hz, 1H), 2.07 (t, J = 7.4 Hz, 2H), 1.48 (h, J= 3.8 Hz, 2H), 1.29 (s, 1H).13C NMR (101 MHz, DMS0-d6) 5 172.36, 167.94, 162.50, 152.44, 143.91. 142.86, 137.22, 137.09, 133.06, 130.95, 130.05, 129.73, 128.20, 127.48, 127.18, 124.33, 118.95, 109.40, 73.65, 60.79, 58.97, 55.63, 55.17, 39.94, 39.73, 39.52, 39.31, 39.10, 38.89, 38.69, 37.99, 34.99, 27.97, 27.81, 24.96. HRMS (ESI) (m / z): calcd for C26H31AuC12N6O4S [M]+, 790.1170; found [M+H], 791.1240 A = -1.007.

[0132] Example 13: Synthesis of AuMacl-biotin Probe.

[0133] To a 25 mL round bottom flask, gold(III) oxime biotin (44.5 mg, 0.056 mmol ). (R,R) -DACH phenyl Trost ligand (38.8 mg, 0.056 mmol) and DCM / MeOH (v / v = 1 : 1) was added and stirred at rt. Progress of the reaction was monitored by using TLC. After 2 h all the ligand was consumed. The solvent was evaporated in vacuo. Flash column chromatography (0- 10 % MeOH in DCM) was used to purify the product. Yield: 30 mg, 58%. Rf = 0.2 (DCM / MeOH, 10: 1). 'H NMR (400 MHz, CDCh) 5 9.53 (s. 1H), 8.65 (s, 2H), 8.51 (d, J= 12.5 Hz. 3H), 8.19 (s, 1H), 7.69 (t, J= 7.5 Hz, 2H). 7.64 - 7.51 (m, 9H). 7.45 (d, J= 7.5 Hz, 2H), 7.34 (dt, J= 15.4, 7.6 Hz, 8H), 7.20 (t, J= 7.6 Hz, 4H), 7.10 (s, 3H), 7.00 (d, J= 20.0 Hz, 6H), 6.82 (d, J= 8.5 Hz, 5H), 6.68 (q, J= 7.0 Hz, 4H), 6.58 (t, J= 7.0 Hz, 4H), 6.34 (s, 3H), 5.99 - 5.88 (m, 5H), 5.28 (d, J= 14.8 Hz, 2H), 4.47 (s, 1H), 4.29 (s, 2H), 4.12 (q, J= 1A Hz, 1H), 3.98 (d, J= 16.1 Hz, 3H), 3.85 (d, J= 16.7 Hz, 2H), 3.42 (d, J = 46.5 Hz,7H), 3.13 (s, 1H), 2.87 (d, J= 12.4 Hz, 2H), 2.27 (s. 3H), 1.93 (s, 5H), 1.70 (s, 7H), 1.55 (d, J = 11.5 Hz, 4H), 1.46 (d, J= 7.2 Hz, 3H), 1.33 (d, J= 3.3 Hz, 4H). ESI-MS m / z (positive mode): calculated for [M-C1J+ C70H71AUCIN8O6P2S 1445.40, found 723.30 ([M-C1J+ / 2).

[0134] Example 14: X-ray crystallography.

[0135] A Bruker D8 Venture dual-source diffractometer was used to collect the low- temperature (90 K) X-ray diffraction data of Au-Macl, and Au-Mac2, and a summary of the cry stallographic information is reported in Tables 1 and 2. Complexes Au-Macl, and Au- Mac2 were dissolved in minimal amount of CHCh, and diethyl ether was used to grow the crystals at room temperature by the vapor diffusion method. All crystals were mounted by using polyisobutene oil on the tip of a fine glass fiber, which was fastened in a copper mounting pin with an electrical solder. Mounted cry stals were placed directly into the cold gas stream of a liquid nitrogen- based cryostat. APEX3 package was used for Lorentz polarization effects such as raw data integration, scaling, merging, and correction. SHELXT and SHELXL were used respectively to determine the space group and refinement. Refinement of nonhydrogen atoms was performed by using anisotropic displacement parameters. Hydrogen atoms were placed at calculated positions and refined by using a riding model with their isotropic displacement parameters (Uiso) set to either 1.2Uiso or 1.5Uiso of the atom to which they were attached. The graphical program SHELXTL-XP was used to draw the ellipsoid plots. All the structures were checked for missed symmetry, twinning, and overall quality with PLATON, an R-tensor before submitting to the Cambridge Structural Database (CSD). Finally, CheckCIF was used for validation of the structures.Table 1: Crystallographic parameters for complexes Au-Macl and Au-Mac2Au-Macl Au-Mac2Formula C57H49AUCI5N3O3P2 C189H204AU3CI5N9O15P6Formula w eight (Mr) 1260.15 3795.57Crystal system Monoclinic HexagonalSpace group C 2 P 6(3)Temperature (K) 90 K 273(2) K a (A) 32.5497(10) 26.3112(13)Z> (A) 16.6566(5) 26.3112(13) c (A) 25.1134(10) 15.5704(12) a (°) 90 90^ (°) 109.088(1) 90 y (°) 90 120Volume (A) 12867.0(8) 9334.9(12)Density (g / cm?) 1.301 1.35 8 2 2.583 2.5355040 3866Data CollectionAbsorption Correction Semi-emperical from equivalents Semi-emperical from equivalentsNumber of Reflections 29015 14269Tmin 0.555 0.667Zmax 0.647 0.746 flint 0.0335 0.0487Refinement fl 1.025 1.015WR20.0568 0.0507TVpar 1318 705Table 2: Selected interatomic distances (A) and bond angles (deg) from the X- ray crystal structure of Au-Macl and Au-Mac2Bond length (A)Au-Macl Au-Mac2Aul - C45 2.0462.038Aul -Cll 2.3554 2.3523Aul - Pl 2.3853 2 3781Aul - P2 2.3883 2.3872Angle ( degree)C45- Aul -Cll 177.117861C45-Aul -P2 90.0791 87Cll-Aul -P2 89.987 14C45-Aul -Pl 92.3291 4CH -Aul -Pl 87.598955P2-Aul-Pl 175.86 17572

[0136] Example 15: Stability of complexes in biologically relevant media.

[0137] Complexes Au-Macl, Au-Mac2, and Au-Mac3 were dissolved in minimal amount of CH3CN and diluted with biologically relevant media DMEM and RPMI, respectively, to make the final concentration of the complexes ~0.7 mM (Img / mL) in 1.0 mL media. At each time point (Oh, 4 h, 8 h, 12 h, and 24 h), 20 pL of the incubated solution (complex and media) was injected into LC / ESI-MS to record the HPLC chromatogram and mass spectrum. The complex mixture was incubated at 37 °C in between injections. The parameters used in the HPLC were as follows: flow rate, 1 mL / min; Z = 280 nm; eluent A = H2O with 0.1 % TFA; Eluent B = CH3CN with 0.05 % HCOOH; Solvent gradient: 0 min (100:0 H2O:CH3CN), 10 min (0: 100 H2O:CH3CN), 15 mm (100:0 H2O:CH3CN).

[0138] Example 16: Reaction with L-Glutathione (GSH).

[0139] 1 mg of compound was dissolved in 200 pL CH3CN and diluted with water to make 500 pL. (1.8 mM). 1.8 mM stock solution of GSH was made by dissolving the GSH in pure water. Equal volume of compound solution and GSH solution was mixed and maintained the complex: GSH = 1 : 1 ratio. Reaction mixtures were incubated at 37 °C in betw een each run. 1 mg of compound w as dissolved in 200 pL CH3CN and diluted with water to make 500 pL. (1.8 mM). 18 mM stock solution of GSH was made by dissolving the GSH in pure water. Equal volume of compound solution and GSH solution w as mixed and maintained the complex: GSH = 1 : 10 ratios. The LC / ESI-MS was recorded at each time point 0 h, 1 h, 4h, 6h, 12h and 24h. The parameters used in the HPLC were as follows: flow rate, 1 mL / min; Z = 280 nm; eluent A = H2O with 0. 1 % TFA; Eluent B = CH3CN with 0.05 % HCOOH; Solvent gradient: 0 min (100:0 H2O:CH3CN), 10 min (0:100 H2O:CH3CN), 15 min (100:0 H2O:CH3CN).

[0140] Example 17: Mouse serum stability test.

[0141] Three (3) female BALB / cJ mice (5 weeks) were received from the Jackson Laboratory (Bar Harbor, ME), and they had an acclimation period of one week. After 9 weeks, serum was collected from the mice. After aliquoting the serum, Au-Macl in DMSO (1 mM) w'as added to each sample and incubated at 37 °C. At varying time points (0, 10, 30, 60, 120, 360. and 1440 min.) methanol was added to each sample and vortexed for 1 minute ai room temperature. And centrifuged to separate the phases. Only supernatant was taken and analyzed by LC / ESI-MS. Two samples were measured at each time point. Peaks of HPLC chromatogram were calculated in each time points. The parameters used in the HPLC were asfollows: flow rate, 1 mL / min; X = 280 nm; eluent A = H2O with 0.1 % TFA; Eluent B = CH3CN with 0.05 % HCOOH; Solvent gradient: 0 min (100:0 H2O:CH3CN), 10 min (0: 100 H2O:CH3CN), 15 mm (100:0 H2O:CH3CN).

[0142] Example 18: Cell culture.

[0143] MDA-MB-231, MDA-MB-468, MDA-MB-436, SUM159 and Hs578T cell lines were purchased from ATCC. MDA-MB-231, MDA-MB-468, and MDA-MB-436 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, 1% penicillin / streptomycin and 1% amphotericin. SUM159 cells were cultured in DMEM / F12 with penicillin / streptomycin / L-glutamine and 10% fetal bovine serum supplemented with 5 pg / ml insulin. Hs578T cells were cultured in high glucose DMEM with 10 pg / ml insulin. penicillin / streptomycin / L-glutamine and 10% fetal bovine serum.

[0144] Ovarian cancer cell lines NIH:OVCAR-3 (OVCAR3) (ATCC® HTB- 161™), OV90 (ATCC® CRL-11732™), SKOV3 (ATCC® HTB-77™), and UWB1.289 (ATCC® CRL- 2945™) were purchased directly from ATCC and absence of mycoplasma was confirmed independently. All cells were maintained as sub confluent monolayers at 37°C, 5% CO2. OVCAR3 and SKOV3 cells were grown in high glucose (4500 mg / L) RPMI 1640 (ATCC) with 0.01 mg / mL bovine insulin (MilliporeSigma) and 20% fetal bovine serum (FBS) (MilliporeSigma). OV90 cells were grown in modified Dulbecco’s Modified Eagle’s Medium (DMEM) (ATCC) containing 4500 mg / L glucose, 4 mM L-glutamine, 1 mM sodium pyruvate, 1500 mg / L sodium bicarbonate supplemented with 10% FBS. UWB1.289 cells were grown in 1 : 1 (v / v) mixture of RPMI- 1640 (with 2 mM L-glutamine) (Lonza) and mammary7epithelial grow th medium (MEGM) (PromoCell) and supplemented with 3% FBS. Platinum-resistant UWB 1.289 cells (UWBl-PlatR) were established from parental cells by incubating in growth media containing 5 pM carboplatin, in recurrent 48-h cycles. Following each 48-h treatment, cells were allowed to recover in platinum-free growth media for 2 to 3 weeks, until typical proliferation resumes, and culture vessel becomes repopulated. A total of 6 cycles were used to establish UWBl-PlatR cells and the stability7of the resistant phenotype was verified for a minimum of 8 passages following 6thtreatment cycle.

[0145] All cells were maintained at 37 °C in a humid atmosphere containing 5% CO2. PBS and trypsin-EDTA used for cell culture maintenance were also purchased from Coming Inc. and used as purchased.

[0146] Example 19: Cell viability assay.

[0147] Various cells (MDA-MB-231, MDA-MB-468, MD-MB-436, SUM159, Hs578T) (3 X 103) were seeded in each well of 96 wells plate. After the cells were incubated overnight at 37 °C, various concentrations of test compounds Au-Macl, Au-Mac2, Au-Mac3, Au-Mac4, Au-Mac5 and ligand (0. 13 - 100 pM) were added and incubated for 72 h (total volume of 200 pL). After 72 h incubation, media was removed and 100 pL of 0.5 mg / mL MTT solution was added to each well in the darkness to protect the MTT from light. Plates were incubated for 3.5 h at 37 °C and MTT solution was removed from each well. 100 pL of DMSO was added to each well and plates were read using plate reader (Absorbance 570 nm). For ovarian cancer cells, treatment conditions were the same except that Cell Titre Gio was used for the assay.

[0148] Example 20: TMRE assay.

[0149] MDA-MB-468 cells were seeded at a density of 5 * 105cells / well in a 6-well plate and allowed to adhere overnight at 37°C. Au-Macl was prepared as a stock in DMSO and treated at final concentrations of 5 and 10 pM after dilution in DMEM. The cells were incubated at 37°C (5% CO2) for 2 h. For a positive control, carbonyl cyanide 3- chlorophenylhydrazone (CCCP) was prepared as a stock in DMSO and added at a final concentration of 100 pM for 1 h. After the incubation time, the cells were trypsinized. pelleted by centrifugation and resuspended in 200 pL TMRE dye solution (200 nM) at room temperature for 20 mins. After incubation time, cells were centrifuged, and the staining solution was removed and replaced with 200 pL of PBS. This was followed by analysis on a flow cytometer with PE channel.

[0150] Example 21: Cell cycle analysis.

[0151] Cells (MDA-MB-468 and MDA-MB-231) (3 x io5) were seeded in each well of a six-well (20 wells) plate. After cells were incubated overnight, 0.5 pM of Au-Macl for 9 wells and 1 pM Au-Macl for 9 wells were added, and 2 wells were kept without adding compounds and incubated at 37 °C. After 6 h incubation, 3 wells per concentration were harvested via trypsinization and centrifuged to form a pellet. The pellet was washed with PBS and transferred to 1.5 mL centrifuge tubes and centrifuged to form a pellet. Then the PBS was decanted, and the pellet was resuspended in 1.0 mL of 70% ethanol in PBS and stored in 4 °C. The same procedure was followed to collect the 12 h and 24 h time points. Atotal of 50 pL of 100 pg / mL RNase solution and 200 pL of 50 pg / mL propidium iodide solution were added to each sample and resuspended and incubated in the ice for 30 mins, and cells were filtered via a strainer. All the samples were analyzed with flow cytometry.

[0152] Example 22: Apoptosis analysis.

[0153] MDAMB468 cells (3 x 105) and MDAMB231 (2 x 105) were seeded in each well of a six-well (12 wells) plate. After the cells were incubated overnight, 0.5 pM of Au- Macl for three wells, 1 pM Au-Macl for three wells were added, and 0.5 pM of DMSO for one well and 200 pM of H2O2 for one well were added and three wells were kept without adding compounds and incubated for 48 h (total volume 2 mL). After 48 h. media was transferred to labeled 15.0 mL centrifuge tubes and were washed with 1.0 mL PBS and transferred to labeled centrifuge tubes. Cells were harvested via trypsinization and transferred to labeled centrifuge tubes. Tubes were centrifuged for 5 min to form a pellet. Pellets were washed with PBS twice and resuspended 500pL annexin binding buffer and transferred to FACS tubes. 5 pL of propidium iodide and 5pL of annexin V-FITC were added to each tube and incubated in the dark for 5 min and analyzed with flow cytometry.

[0154] Example 23: Evaluate the dose dependent effect of cell death suppressing compounds.

[0155] 5000 cells per well (MDA-MB-468) were seeded in the white bottom 96 well plate and allowed to adhere overnight in incubator at 37 °C. Each well was treated with 25 pL of 4 pM (four times of IC50 ) Au-Macl. Plates were treated with serial dilution of 25 pL of Ferrostatin- 1, Necrostatin and zVAD - FMK (100 pM, 50 pM, 25 pM, 12.5 pM, 6.25 pM, 3.13 pM, 1.56 pM, 0 pM). After 24 h incubation, all the plates were kept at room temperature for 30 mins. 60 pL of Cell Titer Gio luminescence solution was added to desired wells. Using the plate reader plates were shaked orbitally for 2 minutes and then plates were allowed to rest for 10 minutes to stabilize. Luminescence w as run scan with 1000 ms integration and 150 ms gain.

[0156] Example 24: Mitochondrial metabolism analysis using Seahorse XF96 analyzer.

[0157] 30,000 cells / well (optimized condition) MDA-MB-468 cells were plated. The cells were seeded the night prior to the experiment with a final volume of 100 pL andincubated overnight at 37°C. Au-Macl was prepared as a stock in DMSO and diluted to a working concentration of 200 LIM with Seahorse XF96 assay buffer and then subsequently serial dilutions were made to achieve multiple concentrations. The assay was performed using a pneumatic injection method of Au-Macl, with the final injection concentrations of 0.1, 1, 3, and 10 pM. This was followed by injection of oligomycin (1.0 pM), FCCP (0.6 pM) and rotenone / antimycin A (0.5 pM). The same procedure was followed for MDA-MB- 231 cells.

[0158] Example 25: Immunoblotting assay.

[0159] The cells were seeded at equal numbers and allowed to adhere and grow to 80% confluence after which the cells were treated with Au-Macl (1 pM) at different time points. At 6 h and 24 h, cells were washed twice with PBS, then RIPA buffer was added, and cells were scraped and collected into 1.5ml centrifuge tubes and kept. The mixture was centrifuged for 15mins at 14,000g and the supernatant was separated from the debris. The cell lysates with equal amount of protein were reconstituted in Laemmli Buffer and heated at 90°C for 10 min. The protein mixture w as then separated by 4-20% SDS-polyacrylamide gel electrophoresis (35 min, 100V) and transferred to PDVF membrane (1 h, 350 mA). The membrane was then blocked with 5% (w / v) bovine serum albumin (BSA) in PBST for 1 h followed by incubation of the membranes with primary antibodies overnight at 4°C. The following day, the membranes were washed three times with PBST and incubated with horseradish peroxi dase-conjugated secondary antibodies (r.t.,1 h) prepared in BSA blocking solution. The membranes were washed with PBST (3x for 5mins) after incubation with secondary antibody. The membranes were placed in pierce enhanced chemiluminescence substrate and visualized with Bio-Rad imager. All antibodies used for this study were purchased from cell signaling technology. The primary antibodies are MFN1, LC3I, LC3II, BECLIN1, OPA1, (CST, #80471S) and 0-actin (CST, #37008).

[0160] Example 26: Pull-down protocol.

[0161] MDA-MB468 cells were plated in DMEM at IxlO6cells per T25 (X6) cell culture flask and allowed to adhere overnight. After 24 h experiment flask was treated with 10 uM Au-Macl -Biotin for 2 h. After 2 h, cells were scraped and collected into a 15 ml tube. Flasks were washed with 1ml of cold PBS and transferred to their corresponding 15 ml tube. Cells were centrifuged, and pellets resuspend with 1 ml cold PBS, and transferred into a 1.5ml tube. This was followed by centrifugation at 2,500 x g(4500 rpm) for 5 min at 4 °C. Decant supernatant, keep pelleted cells. The pellets were resuspended in 0.25 ml of RIPA Buffer, after which the cells were sonicated (30 seconds pulses X 2). The mixture was placed on gently on ice on a shaker to mix for 10 min (total 30 min, keeping on ice, vortex every 10 min). The samples were then centrifuged at -14000 x g (13380 rpm) for 15 min to pellet cell debris. The supernatant solution was separated after centrifugation.

[0162] The biotinylated proteins (cell lysate) were then incubated with streptavidin beads (125 pl) for 30 mins at room temperature. Protein-coated beads were separated with a magnet for 3 mins. The supernatant was removed and the beads were washed with PBS containing 0. 1% BSA. Further wash steps were done in the following sequence: wash with cold PBS (X2), 0.2% Triton xlOO (XI), cold PBS (X3). After the last wash the beads were resuspended in 50 pl of PBS. 20pL of this suspension was mixed with 10 pl of laemmli SDS lysis buffer, vortexed and heated for 15 mins at 80-90 °C. Samples for chemoproteomics were run briefly for 5 mins on the gel, and the gel containing proteins were cut out for proteomic analysis. Samples for HM0X2 IP Western blot was allowed to run fully on the gel for 30 min and previously described immunoblotting protocol followed.

[0163] Example 27: CETSA Protocol.

[0164] Briefly, MDA-MB-468 cells were treated at 10 pM (Au-Macl) for 2 h. After treatment, cells were trypsinized and washed with ice-cold PBS. After washing, the cell pellets were resuspended in ice-cold PBS containing IX protease inhibitor cocktail and aliquoted in 100 pl volume into five tubes (corresponding to 5 different temperature treatments). The tubes were heated at 36, 40, 44, 48, and 52 °C for 3 min and immediately incubated at 25 °C for another 3 min. This was followed by three free-thaw cycles to fully lyse the cells. Cell lysates were next centrifuged at 20,000 x g for 20 mins at 4 °C to remove the insoluble fractions. The supernatant was transferred into new pre-chilled eppendorf tubes labeled appropriately and the protein in the supernatant was denatured by adding Laemmli buffer- SDS Lysis buffer. This was boiled at 90 °C for 5-10 mins and this soluble content was used for western blot detection.

[0165] Example 28: Animals.

[0166] Female, 5 weeks old BALB / c mice were purchased from Charles River Laboratories (Wilmington. MA). All mice were quarantined for 1 week prior to use and kept in micro-isolator cages (four mice per cage) in a temperature- and humidity-controlled environment as per the Division of Laboratory Animal Research (DLAR) of University of Kentucky. All mice were maintained in a pathogen-free environment under the care of DLAR of University of Kentucky. The study was performed in compliance with the NIH guidelines (NIH Publication No. 85-23 Rev. 1985) for the care and use of laboratory animals and all experimental procedures were monitored and approved by the Institutional Animal Care and Use Committee (IACUC) of University of Kentucky (USA).

[0167] Example 29: In vivo efficacy.

[0168] Five (5) female BALB / c mice (5 weeks) were received from Charles River Laboratories (Wilmington, MA), and they had an acclimation period of one week before implanted with 1,000,000 4T1 cells subcutaneously on their flanks. Three days postimplantation, the mice were administered 10 mg / kg Au-Macl intraperitoneally (IP), 0.2 mL / mouse formulated as 1% DMSO, 10% Kolliphor, and 89% PBS. The control group was injected with a PBS solution containing 1% DMSO and 1% Kolliphor. Complex 1 injection, tumor-size / body -weighing measurement were performed three days a week, and mice were euthanized 19 days later. (n=2 for Au-Macl, and n=2 for vehicle control).

[0169] Example 30: Hematoxylin and eosin (H&E) staining.

[0170] The mice used in the in vivo experiment of Au-Macl were sacrificed at day 14 post tumor cells(4Tl) injection. Mice organs (heart, lung, liver, kidney, spleen, and tumor) were fixed in freshly prepared paraformaldehyde (4% in PBS), the fixation time was 24 hours. And those processed for paraffin sectioning. The organs sections of 5 pm were stained with H&E staining and used for histological examination of the organs and tumor. A total of 5 sections per tissue (spanning the full depth of the organ) were examined and photographed using a Nikon Eclipse 55i microscope.

[0171] Example 31: Bio distribution of Au-Macl.

[0172] With four mice inoculated with 4T1 (1,000,000 cells), after 20 days, Au- Macl was administered to mice by IP administration (0.2 mL, 10 mg / kg). Each tissue was obtained by sacrificing the mice (n=2) after 19 days. Obtained tissues were boiled for 5 hoursat 60 °C with 70% nitric acid (0.5 ml) and then boiled again at 60 °C for 10 minutes byadding 35% hydrogen peroxide (0.5 ml). The solution turned yellow and diluted as needed to measure the gold content using a Graphite Furnace Atomic Absorption Spectrometer (GF- AAS). Before measuring all samples, the standard solution curves were measured.

[0173] Example 32: Synthesis and characterization results.

[0174] Using a green C-H activation protocol in water, cyclometalated (CUN) gold(III) complexes were synthesized from commercially available 2-benzoylpyridine, 2- phenoxypyridine, 2-benzylpyridine, 2-phenylpyridine, and benzo [h] quinoline ligands incorporated with HAuCLGFLO. Subsequent associative ligand substitution with the chiral (7?,7?)-DACH phenyl Trost ligand resulted in Au-Mac complexes in >80% yield, affording a novel platform for organometallic macrocyclic Au(III) complexes (FIG. 1A). X-ray single crystals grown from slow diethyl ether diffusion into saturated chloroform solutions of Au- Mac 1 and Au-Mac2 supports the macrocyclic structure (FIG. IB).

[0175] All the complexes were characterized by using 'H-NMR.13C-NMR,31P- NMR, low resolution mass spectrometry (LC / MS), and purity was confirmed by HPLC (>95%) and elemental analysis. A critical bottleneck in metallodrug discovery is the ill- characterized speciation of metal complexes in biological matrices. The solution chemistry and serum stability- of Au-Macl was probed via LC-MS (FIG. 1C-E, FIG. 2A, 2B). In RPMI media, Au-Macl is unaltered (FIG. 2A) and in an equimolar reaction of Au-Macl andglutathione - a dominant biological reductant, Au-Macl remained intact over 24 h (FIG. 3A). To characterize potential Au-Macl intermediates, 1: 10 Au-Macl and L-GSH led to predominantly Au-Macl [m / z 1 104.24] and <5% formation of a reduced Au(I) bisphosphine [m / z 887.20] and concomitant 5-arylated aryl-pyridine motif [m / z 489.14] (FIG. 3B). In mouse serum, -75% of intact Au-Macl is detected over a 24 h period, indicating significant stability of Au-Macl in relevant biological context. For studies with Au-Mac-2, reference is made to FIG. 4A and 4B. For studies with Au-Mac-3, reference is made to FIG. 5A and 5B.

[0176] Example 33: Induction of apoptosis or autophagy in cancer cells.

[0177] Several organogold(IZIII) complexes induce apoptosis or autophagy in cancer cells via ER-stress, mitochondrial dysfunction, and perturb redox homeostasis. Au-Mac recapitulated similar characteristics in cancer cells. Low inhibitory- concentrations (IC50, Table 3) were observed in multiple triple negative breast cancer (TNBC) types includingMDA-MB-231 (human breast cancer), MDA-MB-468 (human breast cancer), MDA-MB-436 (human breast cancer). SUM 159 (human breast cancer), and Hs578T (human breast cancer) 72 h post Au-Macl treatment using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays (FIG. 6A). Under similar conditions, Au-Macl inhibited sensitive and resistant ovarian cancer grow th (FIG. 6B). FIG. 7 is a bar chart summarizing the IC50 values of AU-Mac-1, -2, -3, -4, and -5 in different TNBCs.Table 3: Summary of IC50 values obtained from MTT assays in panel of aggressive TNBC cancer cell lines. 72 h post-treatment.Au-Macl 0.559 ± 0.056 0.636 ± 0.275 0.632 ± 0.118 0.613 ± 0.240 0.486 ± 0.117Au-Mac2 0.426 ± 0.075 0.34 ± 0.225 0.102 ± 0.012 0.400 ± 0.278 0.421 ± 0.152Au-Mac3 1.149 ± 0.053 2.639 ± 0.183 2.052 ± 0.045 1.797 ± 0.181 1.568 ± 0.134Au-Mac4 2.257 ± 0.210 1.066 ± 0.194 2.044 ± 0.206 3.821 ± 0.125 4.520 ± 0.413Au-Mac5 1.971 ± 0.185 1.531 ± 0.115 6.060 ± 0.200 8.329 ± 0.424 6.879 ± 0.454.c0.616 1.92Auranofin± Q Q1 ± 0 20034 56 4 72Cisplatin±o.783 ± 0.180Ligand > 100 > 100 > 100 > 100 > 100

[0178] Example 34: Cell cycle analysis results.

[0179] Overall from 6 h to 24 h treatment of 0.5 pM and 1.0 pM concentrations showed an increase in G1 phase and decrease in G2 and S phases. Early time points (from 6h to 12 h), both concentrations showed an increase in the G2 phase, a decrease in the S phase, and no significant change in the G1 phase (FIG. 8A). MDA-MB-468 cell lines: In 6 h time point, both 0.5 pM and 1.0 pM treatment of the complex showed a slight increase in G1 and G2 phases while showing a slight decrease in the S phase compared to the control. Overall, from 6 h to 24 h treatment of 0.5 pM and 1.0 pM concentrations showed a slight decrease in the G1 and G2 phase and an increase in the S phase (FIG. 8B).

[0180] In MDA-MB-231, time dependent increase were observed in G1 phase of the cell cycle from 6 h to 24 h. In MDA-MB-468, an increase in G1 phase was observed inshorter time (6 h to 12 h). Altogether, cell cycle arrest happened at G1 phase. There are clinically relevant small molecule inhibitors for CDKs 4 and 6 known to arrest cell cycle at G1 phase. CDKs 4 and 6 proteins are responsible for the cellular transition from G1 phase to S phase during cell cycle. Small molecules inhibitors for CDKs 4 &6 are most relevant to treat TNBCs. This data showed us design of this complexes worth to study further in TNBCs.

[0181] Historgrams representing different phases of the cell cycle are provided in FIG. 9A-9E for control, vehicle, and treatment with 0.5 pM and 1.0 pM Au-Macl in MDA- MB-231 and MDA-MB-468.

[0182] Example 35: Apoptosis.

[0183] Toxicity was observed for this class of complexes during cell viability' analysis. It is important to understand the cell death mechanism of this complexes. Apoptosis assay was performed in MDA-MB-231 and MDA-MB-468 cell lines for complex 1. Two different concentrations ( 0.5 and 1 pM) of the complex were treated for 48 h. Observed early stage apoptotic cell population was comparatively low ~6% and -15% in MDA-MB-231 (FIG. 10A) and MDA-MB-468 (FIG. 10B), respectively. FIG. 11A and 11B provide apoptosis analysis plots for Au-Macl in MDA-MB-468 and MDA-MB-231, respectively.

[0184] Example 36: Mitochondria bioenergetics.

[0185] Since Au-Macl is a lipophilic cation and exhibited potent anticancer activity in a broad panel of aggressive cancer cells, it was contemplated that the anticancer mechanism of Au-Macl was via mitochondrial pathways.

[0186] Evidence for disrupted cellular energy homeostasis was confirmed via MitoStress test by Seahorse XF96. Au-Macl impacted the bioenergetics of TNBCs, MDA- MB-468 or MDA-MB-231 cell lines in a concentration-dependent manner, following pneumatic injection of Au-Macl (FIG. 16A, 16B). For MDA-MB-468, extrapolated basal oxy gen consumption rate (OCR) was decreased by 30 % compared to control and maximum OCR was decreased by 50 % at 10 pM Au-Macl compared to control. Importantly, ATP- linked respiration, which is a predictor of energy production was decreased by 75%, and spare respiratory capacity was significantly impacted at 10 pM (FIG. 16C). Similarly, in MDA-MB-231 cells, bioenergetic parameters including basal respiration, maximal respiration. ATP-linked respiration, and spare respiratory capacity were inhibited by Au-Macl in a concentration-dependent manner (FIG. 17). Taken together, Au-Macl induces significant mitochondrial stress.

[0187] Further, the functional consequence of Au-Macl on mitochondrial membrane polarization was evaluated. Significant mitochondrial membrane depolarization (MMP) arose from fluorescence assisted cell sorting (FACS) experiments in MDA-MB-468 cells treated with 5 or 10 pM of Au-Macl or 100 pM CCCP along with TMRE MMP marker (FIG. 12A, 12B). There was a dose-dependent decrease in fluorescence signals for Au-Macl treated cells compared to CCCP-treated and untreated controls, indicative of strong mitochondrial membrane depolarization by Au-Macl. These studies prompted a detailed study into the plausible molecular target and mechanism of action of Au-Macl. Thus, it is contemplated that a biotinylated probe of Au-Macl will facilitate target identification in the context of cancer.

[0188] Example 37: Development of a Biotinylated-AuMacl Probe.

[0189] Follow ing the identification of Au-Macl as an inducer of mitochondrial dysfunction, studies were designed to elucidate its targets by developing a chemical biology probe for a deeper understanding of its mechanism. In this regard, biotin was introduced on the oxime-functionalized cyclometalated Au(III) dichloride precursor via a peptide linkage. After which, chelation with phosphine donor ligands was performed and purified via flash chromatography to obtain the cationic Au-Macl -biotin probe (Example 9, Scheme 3), which was fully characterized by NMR spectroscopy and LC-MS.

[0190] Example 38: Chemoproteomic profiling

[0191] To identify the molecular target of Au-Macl, live MDA-MB-468 cells were treated with Au-Macl -biotin at 10 pM for 2 h. The treated cells were lysed, and the lysates were incubated with streptavidin magnetic beads to pull down the biotinylated proteomes. Isolated streptavidin beads from both treated or untreated cells w ere loaded unto SDS-PAGE and run for 5 min. The gel bands w ere cut and prepared for try ptic digestion and LC-MS / MS analysis of the enriched peptides to profile the targets (FIG. 13A). Proteins not found in the control group while showing a high abundance in the probe treatment group were identified as the possible targets. Volcanic plot showed HM0X2 as the highly ranked differentially expressed protein (FIG. 13B).

[0192] Additional insight into the protein pathway interactome associated with identified HM0X2 using STRING analysis, a web-based platform for dissecting protein interaction showed heme metabolism and mitochondrial localization (FIG. 13C). Particularly, proteins clustered within heme metabolic process included BLVRA, BLVRA, and HM0X1. Related mitochondrial proteins involved in iron processing were identified as ferrochelatase, FECH and cytochrome c oxidase 10 (COX) assembly protein, which catalyzes the famesylation of the C2 vinyl group of heme, resulting in the transformation of protoheme (heme B) to heme O. It is becoming increasingly clear that perturbing cellular homeostasis has wide metabolic implications leading to effective therapies. Examination of enriched proteins via pull-down studies showed HM0X2 abundance using HMOX2-specific antibodyin an immunoblotting assay as depicted in FIG. 13D.

[0193] Example 39: Validation of HMOX2 Target engagement by Au-Macl.

[0194] Intracellular protein target engagement to verily the interaction of Au-Macl with HM0X2 in MDA-MB-468 cells was performed by the cellular thermal shift assay (CETSA). Based on the phenomenon that ligand-protein binding affects protein stability, the Nordlund’s group developed CETSA to directly monitor target engagement inside cells. Thermally induced unfolding experiments provide distinct melting curves for a protein, and these melting curves typically shift to a higher temperature when a ligand binds to and stabilize the target protein. Using CETSA to examine whether Au-Macl binds to endogenous HM0X2, MDA-MB-468 cells treated with or without Au-Macl (10 pM) for 2 h were collected, followed by heating at 36. 40, 44, 48, and 52 °C to denature and precipitate HM0X2 protein, cell lysis, removal of cell debris and aggregates by centrifugation, and finally detection of remaining thermostable HM0X2 protein by Western blot. The HM0X2 aggregation temperatures without Au-Macl were determined to be 44-52 °C but shifted to 48-52 °C after treatment with Au-Macl (FIG. 14A-14C). Further confirmation by isothermal dose response CETSA supported intracellular engagement of HM0X2 by Au-Mac 1 as higher concentrations at 10 pM stabilized the protein at the melting temperature of 48 °C (FIG. 14D). Overall, a chemical biology approach was used to identify and validate a unique Au(III) target.

[0195] HM0X2 modulation can influence oxidative stress and mitochondrial membrane depolarization, which promotes degradation of mitochondrial membrane proteins such as OPA1 and MFN 1&2 via mitophagy. Au agents that rapidly and irreversibly degradefusion present opportunities to uncover new mechanisms, generate tool compounds, and therapeutics for disease. Biochemical hallmarks that characterize fusion protein degradation include: i) depletion of mitochondrial inner membrane fusion proteins, ii) autophagy / mitophagy activation, and iii) modulation of cellular energy homeostasis. Orchestration of these factors in concert suggest mitochondrial dynamics fusion protein degradation in cells. Protein expression of the transmembrane GTPases 0PA1 and MFN1, a mitofusin that regulates mitochondrial dynamics and cellular homeostasis was assessed via immunoblotting and found a robust depletion of both proteins in MDA-MB-468 cells by 6 - 24 h following 1 pM treatment (FIG. 15A,15B). OPA1 is a fusion protein within the inner mitochondrial membrane, which has been implicated in several cancer mechanisms including cell proliferation, cytochrome C release and angiogenesis. Furthermore, since disruption of mitochondrial dynamics can lead to cell death, it was contemplated that Au-Mac 1 can induce both mitophagy / autophagy and apoptosis in TNBC. This was tested using immunoblotting to monitor the expression of autophagy' markers (light chain protein 3) LC3-I and LC3-II as well as the mitophagy marker Beclin 1 (FIG. 15C, 15D). Au-Mac 1 -induced expression of LC3-II / LC3-I represents an autophagic response. The autophagy protein Beclin 1 is critical in autophagosome formation, mitofusin degradation and mitophagy initiation. Evidence for time-dependent Beclin 1 expression upon 1 pM Au-Macl treatment suggest the onset of autophagy / mitophagy' as an important cell death pathway (FIG. 15D). To evaluate the extent of apoptosis induced by Au-Macl. Annexin V / PI dual staining assay was used to detect cells undergoing early and late-stage apoptosis. A significant population of cells treated with Au- Macl undergo apoptosis (FIG. 15E). Moreover, the dose-dependent effects of cell death suppressors (Ferrostatin -1: Ferroptosis inhibitor, Necrostatin-1: Necrosis inhibitor, and zVAD: Apoptosis inhibitor) on MDA-MB-468 cells treated with lethal concentration (4 pM) of Au-Macl were analyzed using Cell Titer Gio assay (FIG. 15F). The cell survival plot revealed that zVAD inhibits cell death, indicating apoptosis as a dominant cell death pathway.

[0196] Example 40: In vivo efficacy and bio distribution.

[0197] The in vivo efficacy of Au-Macl was assessed in a BALB / c mouse model. A syngeneic model was established via subcutaneous implantation of 4T1 cells. 4T1 is a metastatic TNBC of mouse origin, that provides an immunocompetent background to study drug efficacy. Au-Macl was administered at 10 mg / kg via intraperitoneal (IP) injection 3times per week (FIG. 18A). A significant decrease was observed in the tumor volume after 12 days of treatment and there was no significant change in the body weight between treated and control mice, indicative of enhanced in vivo efficacy and tolerability of Au-Macl (FIG. 18B, 18C)

[0198] Further, tissue biodistribution of Au-Macl was analyzed in different organs of treated mice. Drug biodistribution provides information of complex accumulation and elucidates drug metabolism and potential side effects. The highest content of gold was found in the kidney and liver (FIG. 18E), suggesting a possible clearance pathway via these organs. Hematoxylin and eosin staining was performed to evaluate the cellularity of cancer tissue and preliminary’ toxicity in different organs. H & E staining revealed that the tumor proliferation was reduced in treated tumor tissue and other tissues did not show any difference in treated vs control tissue (FIG. 18E). FIG. 19 includes a standard curve for the biodistribution analysis. Overall, Au-Macl inhibits metastatic 4T1 TNBC in mice with significant tolerability and reduced adverse effects.

[0199] Example 41: Au-Macl inhibits TNBC and TNBC CSCs growth in vitro.

[0200] All cell lines were purchased from the American Type Culture Collection ATCC and grown in a humidified incubator at 37 °C with 5% CO2. MDA-MB-468 and SUM159 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, 1% penicillin / streptomycin and 1% amphotericin. All the supplements used were purchased from Coming Inc. PBS and trypsin-EDTA used for cell culture maintenance were also purchased from Coming Inc. and used as purchased.

[0201] Cell viability’ was assessed using the MTT assay. The TNBC cells were seeded in 96-well plates (3000 cells / well) in their respective grow th media and allowed to adhere for 24h. Au-Macl was prepared as a stock (ImM) in DMSO / media mixture. The compound was added at increasing concentrations with a highest concentration and seven serial dilutions (l / 3x). The cells were placed in the incubator at 37 °C with 5% CO2 for 72h. After incubation time, the medium w as removed and MTT solution w as added at a final concentration of 0.5mg / ml to the wells followed by incubation for 4h. After incubation, the MTT reagent was removed and 100 pl of DMSO was added to dissolve the formazan crystals. The absorbance was measured at 570nm using the Biotek plate reader (X = 570 nm).The experiment was performed in triplicate and the IC50 values were calculated using GraphPad Prism software. Mean ± s.e.m. (n = 3).

[0202] For spheroid formation, TNBC cells were trypsinized, pelleted and resuspended as single cell suspension in MammoCult Human Medium (StemCell Technologies) supplemented with Heparin (4 qg / ml, StemCellTechnologies) and Hydrocortisone (0.48 pg / ml. StemCell Technologies). 4000 cells / mL per well were plated in 24-well ultra-low attachment plates. Cells were treated the following day (after 24 hrs) as control and 1 pM Au-Macl for 5 days. Spheres were photographed at the end of the experiment.

[0203] For cell sorting, SUMI 59 cells were trypsinized, pelleted and cells were resuspended in PBS containing 2% FBS at concentration of l x 106cells in 100 ul and incubated with 20 ul of ESA-FITC , CD44-APC, and CD24-PE as required. This was incubated for 20 mins on ice after which the cells were washed twice with PBS and resuspended in 300ul of PBS containing 2% FBS before flow cytometry analysis. CD44+I CD24" cells were sorted into a separate tubes in put in T25 culture flask to grow.

[0204] In a 72h MTT assay, Au-Macl effectively inhibited the growth of TNBC cells, MDA-MB-468 and SUM159 in vitro (FIG. 20A). Given the observed efficacy of Au- Macl against the TNBC cells the efficacy against the TNBC CSCs population was also of interest. First, the mammosphere formation assay (MFA) was peformed, which enables evaluation of drug efficacy against CSCs in vitro. Au-Macl inhibited mammosphere formation in MDA-MB-468 and SUM159 cells (FIG. 20B, 20C). To validate the effect of Au-Macl on TNBC -CSCs, cell sorting was performed to isolate CD44 / CD24’ stem cell population from the whole SUM 159 cell population (FIG. 20D). Au-Macl was equally effective in inhibiting the proliferation of CD447CD24" cell population in a 72h MTT assay (FIG. 20E). The MFA was performed using the sorted CD447CD24' cells and it showed that mammosphere formation was inhibited upon exposure to Au-Macl (FIG. 20F) Given the current lack of chemotherapeutic agents in the clinic capable of eliminating CSCs, these results highlight Au-Macl as a promising agent for TNBC chemotherapy.

[0205] Example 42: Au-Macl inhibits mitochondrial respiration in TNBC and TNBC CSCs.

[0206] TNBC cells were plated at 30,000 cells / well for the Seahorse XF96 experiments. The cells were plated a day before the experiment in a 100 pL volume per well and incubated overnight at 37 °C. Au-Macl was prepared as a ImM stock in DMSO and phenol red free DMEM and diluted to a working concentration with Seahorse XF96 assay buffer. For 12h pretreatment, the cells were incubated with Au-Macl at 0.5 pM and 2 pM for 12 h. This was followed by injection of oligomycin (1.5 pM), FCCP (0.6 pM) and rotenone / antimycin A (0.5 pM). The metabolic parameters are extrapolated based on readings gotten from a minimum of six wells.

[0207] TNBC cells are known to exhibit metabolic heterogeneity with dependence on both glycolysis and mitochondria oxidative phosphorylation (OXPHOS). Moreover, small molecule anticancer agents that interfere with mitochondrial respiration have shown potency against different TNBC models in different pre-clinical studies. Also, it has been reported that mitochondria metabolism is important for the survival of CSCs and supports drug resistance in CSCs. Based on precedent from previous studies that lipophilic cationic gold complexes are capable of localizing in the mitochondrial of cancer cells to disrupt mitochondrial physiological processes, the effect of Au-Macl (which is also a lipophilic cationic complex) was examined on mitochondrial respiration in both TNBC cells and TNBC isolated CD44+ / CD24‘ stem cell population. Mitochondrial respiration in TNBC and isolated CD44+ / CD24‘ stem cell population was significantly inhibited in a concentration dependent manner at 0.5 and 2 pM as quantified by extrapolated parameters like maximal respiration and ATP production (FIG. 21A-21D).

[0208] Mitochondrial biogenesis is transcriptionally regulated by the peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-la). Studies have shown that high PGC-la expression level in cancer cells is associated with dependence on mitochondria respiration. Additionally, PGC-la has been shown to support tumor metastasis in TNBC via mitochondria OXPHOS. Similarly, in CSCs it has been reported that mitochondrial biogenesis supports the survival of breast cancer cells. Another master transcription factor involved in the regulation of mitochondrial biogenesis is the c-Myc protooncogene4. Several mitochondria genes are under regulation by c-MYC and in tumor progression, c-myc elevates mitochondria respiration to promote proliferation of tumor cells4. The effect of Au-Macl on the expression of PGC-la and c-MYC was examined. Interestingly, Au-Macl caused a rapid decrease in the expression levels of PGC-la and c-MYC as early as 6 h at 1 pM concentration in MDA-MB-468 and SUM159 cells (FIG. 21E, 21F). Depletion of c-MYC by Au-Macl was similarly observed in MDA-MB-436 and MDA- MB-231 TNBC cell lines.

[0209] Although p62 gene is associated with regulating autophagy, recent studies have also shown that p62 plays a role in mitochondrial energy’ metabolism. A knock down of p62 in human neuroblastoma cell line (SH-SY5Y) was previously performed and this revealed that mitochondrial membrane potential is compromised in addition to decreased mitochondrial respiration upon p62 depletion. Interestingly, p62 has also been implicated in the development of breast cancer. It was previously found that p62 is important in the regulation of breast cancer stem cells as it is highly expressed in the CSCs. Surprisingly, a positive correlation between p62 and MYC expression levels was also previously found and it has been previously reported that knock down of p62 caused decreased MY C expression which was restored by p62 overexpression. Such findings unveiled p62 as an upstream regulator of c-myc mRNA. It should also be noted that c-myc itself is known as an important regulator of sternness in CSCs as it contributes to the maintenance of CSCs properties. Based on the effect of Au-Macl on isolated TNBC CD44+ / CD24‘ TNBC - CSCs, Myc depletion by Au-Macl, and recent evidence of a regulatory' role of p62 gene on MYC expression, the effect of Au-Macl on p62 expression was examined. Interestingly, p62 expression is decreased in SUM159 and MDA-MB468 cells treated with 1 pM Au-Macl (FIG. 21E, 21F).

[0210] Example 43: Target validation studies shows regulatory effect of HMOX2 on p62 and c-MYC.

[0211] TNBC cells were seeded at equal density' and allowed to adhere and grow to 80% confluence after which the cells were treated with Au-Macl at the indicated concentrations and time. After treatment time, cells were washed twice with PBS, then Laemmli was added, and cells were scraped and collected into 1.5ml centrifuge tubes. The samples were heated at 90°C for 5 min. The protein mixture was then separated by 4-20% SDS-polyacrylamide gel electrophoresis (35 min, 100V) and transferred to PDVF membrane (Ihr, 350mA). The membrane was then blocked with 5% (w / v) bovine serum albumin (BSA) in TBST for 1 h followed by incubation of the membranes overnight with primary antibodies overnight at 4°C. The following day, the membranes were washed three times with TBST (IX) and incubated with horseradish peroxidase-conjugated secondary antibodies (r.t, Ih) prepared in BSA blocking solution. The membranes were washed with TBST (X3 for 5mins)after incubation with secondary' antibody. The membranes were placed in pierce enhanced chemiluminescence substrate and visualized with Bio-Rad imager.

[0212] Target identification studies were conducted with biotinylated Au-Macl which led to identification of HM0X2 as a potential protein target of Au-Macl. The next step here was to answer the question as to if genetic perturbation of HM0X2 protein will recapitulate Au-Macl induced inhibition of p62 and MYC. siRNA knockdown of HM0X2 was carried out and p62 and MYC levels were found to be depleted upon HM0X2 knockdown in MDA-MB-468 (FIG. 22A) and SUM159 cells (FIG. 22B). This represents an interesting finding as it show s a potentially regulatory effect of HM0X2 on p62 and MYC genes.

[0213] Example 44: Au-Macl inhibits growth of human TNBC.

[0214] For the experiments using the subcutaneous xenograft mouse model of TNBC, 2 * 106SUM159 cells were inoculated orthotopically to the mammary fat pad of 8-week-old athymic nude mice (Jackson lab, USA). Once the tumors were established at 50 mm3, mice were randomized for treatment. Intraperitoneal administrations of vehicle and therapeutic agents were performed in a volume of 100 pl.

[0215] The therapeutic efficacy of Au-Macl (LR13) was performed by randomization according to weight and tumor burden at treatment onset. Au-Macl w as administered intraperitoneally at a dose of 5 mg / kg three times per week. To evaluate the therapeutic tolerance of Au-Macl after systemic administration, the body weight of mice was measured every other day. After the study, tissues were harvested and fixed for histological analysis or flash frozen for graphite furnace atomic absorption spectroscopy (GF- AAS) to assess gold biodistribution.

[0216] To determine the safety' and therapeutic potential of Au-Macl as a single agent administered in vivo, an orthotopic xenograft mouse model was used of human SUMI 59 cells, which is a mesenchymal TNBC with enriched stem cell population. Upon establishment of the tumors (average tumor size of 50 mm3), mice were randomized for systemic administration with Au-Macl, or vehicle control, which were administered three times a w eek by intraperitoneal administration. During the experiments, Au-Macl treated mice showed reduced tumor growth as indicated by tumor volume measurements (FIG. 23A,23C). Furthermore, the dosing schedule of Au-Mac 1 was well tolerated at the dosing schedule used as monitored through changes in body weight (FIG. 23B).

[0217] Example 45: Methods for Ovarian Cancer Studies.

[0218] Cell culture. All cell lines used for this study were purchased from the American Type Culture Collection ATCC and grown in a humidified incubator at 37 °C with 5% CO2. OVCAR3 cells were grown in RPMI-1640 supplemented with 20% FBS , 10 pg / ml insulin, 1% penicillin / streptomycin and 1% amphotericin B UWB1.289 and UWB1.289- PlalR cells were maintained in 1: 1 (v : v) RPMI-1640:MEGM supplemented with 3% FBS, 1% penicillin / streptomycin and 1% amphotericin B. OV90 cells were maintained in 1: 1 (wv) MCDB I 05 : Medium 199 supplemented with 15% FBS, 1 % penicillin / streptomycin and 1 % amphotericin B. RPMI-1640, penicillin / streptomycin and amphotericin B, were purchased from Coming Inc. MEGM was purchased from Lonza (Lonza’s Mammary Epithelial Cell Growth Medium (MEGM) supplemented with Lonza’s MEGM bullet kit containing. Bovine Pituitary Extract (BPE), human epidermal growth factor (hEGF), insulin, hydrocortisone and Gentamicin- Amphotericin (GA- 1000)). Insulin solution (from bovine pancreas), MCDB105 and Medium 199 were purchased from Sigma Aldrich. FBS was purchased from Atlasbio. PBS and trypsin-EDTA used for cell culture maintenance were also purchased from Coming Inc. and used as purchased.

[0219] Chemistry. All solvents used for this study w ere of ACS grade and used as purchased from Pharmco-Aaper without further purification. All reactions and experiment were carried out in air under standard atmospheric condition unless indicated differently. Nuclear magnetic resonance (NMR) spectra were obtained at room temperature on Bruker Avance NEO 400 MHz spectrometer and 500 MHzJEOL ECZr. Chemical shift and coupling constant are represented in ppm and Hz respectively . The multiplicity of the signals is represented as singlet (s), doublet (d), m (multiplet) and broad (br). Au-Macl was synthesized as previously described. Dichloro (2-benzoylpyridine)gold(III) 1 was synthesized as previously described. 2 w as synthesized as previously described. The purity of the synthesized compounds were determined by reversed-phase HPLC (RP-HPLC) (Agilent Technologies 1100 series HPLC instrument with an Agilent Phase Eclipse Plus C18 column (4.6 mm x 100 mm; 3.5 pm particle size)).

[0220] To a scintillation vial was added compound 2 (50 mg, 0.099 mmol,l equiv) followed by (R,R)-DACH phenyl Trost ligand (68.4 mg, 0.099 mmol, 1 equiv), and 6 mL of MeOH / DCM (v / v = 1: 1). This mixture was stirred for 2h:30mins at room temperature and monitored by TLC. A light-yellow clear solution was formed. The solvent was evaporated with the rotovap and the dried product was redissolved in a small amount of DCM and precipitated with diethyl ether. The dried product from the precipitation was purified using the flash column.XH NMR (500 MHz. Chloroform-c / ) 5 8.94 (d, J= 27.6 Hz. 1H), 8.48 (s, 1H), 8.33 (d, J = 4.4 Hz, 1H), 8.28 (dd, J= 8.0, 1.4 Hz, 1H), 7.67 - 7.42 (m, 7H), 7.33 (dd, J = 27.8, 7.3 Hz, 3H), 7.25 - 7.13 (m, 4H), 7.11 - 7.03 (m, 1H), 7.00 (d, J= 8.5 Hz, 2H), 6.95 - 6.82 (m, 4H), 6.72 (tdd, J= 20.2, 10.2, 4.6 Hz, 4H), 6.60 (dd, J= 18.0, 7.7 Hz, 1H), 6.38 - 6.24 (m, 1H), 5.29 (d, J= 11.8 Hz, 1H), 3.94 (s. 1H), 2.05 - 1.94 (m, 2H), 1.66 (s, 4H).31P NMR (202 MHz, Chloroform-D) 5 36.71, 36.33.

[0221] Cell viability assay. Cell viability was assessed using the MTT assay. The Ovarian cancer cells were seeded in 96-well plates (3000 cells / well) in their respective growth media and allowed to adhere for 24h. Au-Mac 1 was prepared as a stock (ImM) in DMSO / media mixture. The compound was added at increasing concentrations with a highest concentration and seven serial dilutions (l / 3x). The cells were placed in the incubator at 37 °C with 5% CO2 for 72h. After incubation time, the medium was removed and MTT solution was added at a final concentration of 0.5mg / ml to the wells followed by incubation for 4h. After incubation, the MTT reagent was removed and 100 pl of DMSO was added to dissolve the formazan crystals. The absorbance was measured at 570nm using the Biotek plate reader (X = 570 nm). The experiment was performed in triplicate and the IC50 values were calculated using GraphPad Prism software. Mean ± s.e.m. (n = 3).

[0222] Colony formation assay. UWB 1.289 and UWB1.289-PlatR cells were plated in 6 wells plate at 2000 cells per well. Cells were cultured with and without compound (Au- Macl) for 10 days. After incubation period, the cells were fixed with 4% paraformaldehyde for 20mins and stained with crystal violet for 15mins and the number of colonies w ere counted.

[0223] Spheroid formation assay. UWB1.289 and UWB1.289-Carb R cells were trypsinized, pelleted and resuspended as single cell suspension in MammoCult Human Medium (StemCell Technologies) supplemented with Heparin (4 pg / ml, StemCellTechnologies) and Hydrocortisone (0.48 pg / ml, StemCell Technologies). 4000cells / mL per well were plated in 24-well ultra-low attachment plates. Cells were treated the following day (after 24 hrs) as control and 1 pM Au-Macl for 5 days. Spheres were photographed at the end of the experiment.

[0224] Mitochondrial membrane potential (TMRE). UWB 1.289 and UWB 1.289- PlatR cells were plated in six wells plate at 500,000 cells per well and allowed to adhere overnight. Cells were treated with Au-Macl for 2 h at 5 pM and 10 pM. After incubation time with test compound, cells were trypsinized and pelleted by centrifugation. Pellets were washed with PBS and resuspended in TMRE dye solution (200 nM, 200 pL). This was transferred into FACS tube and incubated for 20mins followed by flow cytometry analysis.

[0225] Mitochondrial ROS (mtROS). UWB 1 .289 and UWB1 289-PlatR cells were plated in six wells plate at 300,000 cells per well and allowed to adhere overnight. Cells were treated with LR-13 for 2 h at 2 pM and 5 pM. After incubation time with test compound, cells were trypsinized and pelleted by centrifugation. Pellets were washed with PBS and resuspended in Mitosox dye solution (5pM, 150 pL). This was transferred into FACS tube and incubated for 20mins followed by flow cytometry analysis.

[0226] Fluorescence visualization of Au-Macl in cells. UWB1.289 cells were seeded at 3X105cells / ml on 35 mm glass-bottom Petri dishes and incubated overnight in appropriate grow th medium at 37°C. After 24h, media was removed and cells were washed with PBS twice, followed by treatment w ith Au-Macl -Alkyne at 20 pM for 1 h, the control dish was left untreated. After treatment with test compound, cells were washed three times with PBS followed by treatment with mitotracker red (200 nM) for 45 mins after which the cells were washed three times and fixed with 4% PFA for 20 mins. After fixing, cells were washed three times and permeabilized with 0.2 % triton -X for 1 h at room temperature. Cells were washed three times with PBS after permeabilization. In a 15 ml centrifuge tube, azide fluorescein (final concentration of 100 pM) and amino guanidine (final concentration of 1 mM) were mixed in PBS, (total reaction volume of 3ml) placed on ice. To this mixture was added CuSC>4 and THPTA (molar ratio of 1 :5). A stock solution of sodium ascorbate (100 mM) w as prepared fresh and added to the mixture for a final ascorbate concentration of (2.5 mM) in the mixture. The reaction mixture was placed on ice for 10 mins after which it was added to the cells for 30 mins (cells w ere placed on ice). After Ih, cells were washed three times and the Hoechst dye was added for 15 mins to stain the nuclei. Cells were washed with PBS andmounted on the Nikon AIR confocal microscope with 60X oil objective Images were analyzed with NIS viewer.

[0227] Bioenergetics measurement with Seahorse XF96 analysis. Ovarian cancer cells were plated at 30,000 cells / well for the Seahorse XF96 experiments. The cells were plated a day before the experiment in a 100 pL volume per well and incubated overnight at 37 °C. Au-Macl was prepared as a ImM stock in DMSO and phenol red free DMEM and diluted to a working concentration with Seahorse XF96 assay buffer. The assay involved a pneumatic injection method of Au-Macl, with the final injection concentrations of 1 pM, 5 pM and 10 pM. For 12h pretreatment, the cells were incubated with Au-Macl at 0.5 pM, 1 pM, 2 pM. and 4 pM for 12 h. This was followed by injection of oligomycin (1.5 pM), FCCP (0.6 pM) and rotenone / antimycin A (0.5 pM). The metabolic parameters are extrapolated based on readings gotten from a minimum of six wells.

[0228] Immunoblotting. UWB1.289 and UWB1.289-Plat R cells were seeded at equal density and allowed to adhere and grow to 80% confluence after which the cells were treated with Au-Macl at the indicated concentrations and time. After treatment time, cells were washed twice with PBS, then Laemmli was added, and cells were scraped and collected into 1.5ml centrifuge tubes. The samples were heated at 90°C for 5 min. The protein mixture was then separated by 4-20% SDS-polyacrylamide gel electrophoresis (35 min. 100V) and transferred to PDVF membrane (Ihr, 350mA). The membrane was then blocked with 5% (w / v) bovine serum albumin (BSA) in TBST for 1 h followed by incubation of the membranes overnight with primary' antibodies overnight at 4°C. The following day, the membranes were washed three times with TBST (IX) and incubated with horseradish peroxidase-conjugated secondary antibodies (r.t, Ih) prepared in BSA blocking solution. The membranes were washed with TBST (X3 for 5mins) after incubation with secondary' antibody. The membranes were placed in pierce enhanced chemiluminescence substrate and visualized with Bio-Rad imager. The primary antibodies are OPA1 (CST, #80471 S), MFN1 (CST, # 14739S), c-myc (CST, # 13987T), PINK 1, (CST, #6946S), Parkin (sc-32282). Ubiquitin (sc-8017), Phospho-Ubiquitin (Ser65) (CST, #628028), ATF4, (CST, #1 1815S) and P-actin (CST, #3700S).

[0229] Transmission electron microscopy. UWB1.289 control and Au-Macl treated cells (10 pM) were processed for TEM and imaged at the University of Kentucky College of Engineering Electron Microscopy Center.

[0230] Immunoprecipitation. UWB1.289 and Carb R cells were grown and incubated with the test compound (Au-Macl) for 24hrs at concentration of 1 pM. Lysates were prepared using RIP A lysis buffer. To the lysates, MFN1 antibody was added and incubated overnight at 4 °C. After overnight incubation with MFN1 antibody, protein A / G agarose bead (Santacruz Biotechnology7) was added to control and 24hrs samples and samples were rotated at 4 °C for 2 h. After incubation, sample was centrifuged for 5 mins and the supernatant separated from the beads. The beads were washed 3 times with ice cold RIPA buffer (centrifuging at 2500 rpm for 5 mins in-between each washes). After the wash step is completed, the beads were resuspended in SDS lysis buffer (Laemmli) and boiled for 5 mins at 90 °C, centrifuged briefly at 2500 rpm for 2 mins to separate the bead from the supernatant, after which the samples were subjected to westemblot with K48 polyubiquitin antibody.

[0231] Example 46: Au-Macl inhibits proliferation of HGSOC.

[0232] Eight structurally diverse gold-based complexes were screened across a panel of HGSOC (UWB 1.289, OVCAR3, SKOV3 and OV90) using the MTT assay. Based on the current clinical challenge of platinum resistance in ovarian cancer patients, a platinum resistant variant of UWB1.289 referred to as UWB1.289-PlatR was included in the screen. A macrocyclic complex was determined to be most potent across all cell lines tested with ICso values within the range of (0.3 - 0.7pM) after 72 h of incubation. To further examine the antiproliferative effect of Au-Macl on OC cells, colony formation assays were performed. The results show that colony formation was significantly inhibited by Au-Macl after treatment for 7 days (FIG. 24B). To evaluate the translational utility of Au-Macl for ovarian cancer chemotherapy, the 3D spheroid formation assay was performed, which is a better model to recapitulate the complex in vivo tumor environment. Au-Macl significantly inhibited spheroid formation in UWB1.289 and UWB1.289-PlatR at 1 pM (FIG. 24C-24D), highlighting the therapeutic utility of Au-Macl for OC treatment.

[0233] Example 47: Au-Macl inhibits mitochondrial fusion in OC.

[0234] Next, the mechanism by which Au-Macl inhibits ovarian cancer was investigated. Based on the lipophilic cationic character of Au-Macl, it was contemplated that it could accumulate in the mitochondria of cancer cells. This was tested by examining the effects of Au-Macl on mitochondrial membrane potential (MMP). MMP was measured usingthe tetramethylrhodamine ethyl ester (TMRE) dye; a cationic fluorescent dye that accumulates in active mitochondria. However, when the mitochondria are depolarized, the TMRE dye is poorly retained. A concentration-dependent decrease in TMRE fluorescence intensity was observed in Au-Mac 1 treated cells, indicating depolarization of the mitochondria membrane by Au-Mac 1 (FIG. 25A). Given the relationship between MMP depolarization and mitochondria ROS (mtROS) production. mtROS levels were examined in Au-Macl treated cells using the mitochondria-specific MitoSOX red dye. A significant increase in the mean fluorescence intensity of the mitoSOX dye was observed, which indicates greater mtROS production in Au-Macl treated cells compared to the control cells (FIG. 25B)

[0235] To further confirm that Au-Macl targets the mitochondria of ovarian cancer cells, Au-Macl localization was examined via click chemistry. Au-Macl was modified to form a probe (Au-Macl -alkyne-probe) by incorporating an alkyne handle to enable fluorescence visualization via copper-catalyzed azide-alkyne cycloaddition (CuACC) click chemistry. Colocalization of Au-Macl -AP with a mitochondria-specific dye MitoTracker Red CM-HjXRos (MTR), was demonstrated using fluorescence microscopy (FIG. 25C)

[0236] Recent studies have shown that depolarization of the mitochondria membrane may perturb mitochondria dynamics. To further gain insights into the mechanism of Au- Macl induced cell death in ovarian cancer, the effect of Au-Macl on mitochondria structure was examined. Interestingly, Au-Macl induced significant decrease in the expression of mitochondria fusion protein MFN1 and degradation of an isoform of OP Al at a concentration of IpM within 24 h treatment in both UWB 1.289 and the platinum resistant variant (UWBI.289-Plat R) (FIG. 25D). Inhibition of the fusion proteins consequently caused fragmentation of the mitochondria as assessed by transmission electron microscopy (FIG. 25E)

[0237] Example 48: Au-Macl inhibits mitochondrial respiration in OC.

[0238] Pharmacologic induction of mitochondrial membrane depolarization and disruption of mitochondria dynamics have implications on mitochondrial metabolism. The effect of Au-Macl on mitochondrial oxidative phosphorylation (OXPHOS) was examined using the Mho stress test assay. Short exposure of UWB 1.289, UWB1.289-PlatR, OVCAR3, SK.OV3 and OV90 cells to Au-Macl caused rapid decrease in ATP production, while basaland maximal respiration were unaffected (FIG. 26A-26D). A 12 h incubation with Au-Mac 1 resulted in a dose dependent inhibition of mitochondria respiration as quantified by parameters like basal respiration, maximal respiration, and ATP production (FIG. 26E-26F).

[0239] The MYC proto-oncogene has been implicated as a transcriptional regulator of mitochondria related processes including mitochondria dynamics, mitochondria biogenesis, and metabolism. UWB1.289 and UWB1.289-Plat R cells were treated w’lth Au-Macl at IpM and examined MYC expression The results show that MYC expression is decreased in 6 h and sustained up to 24 h of treatment with Au-Macl (FIG. 26G). Further, the results show that the activating transcription factor 4 (ATF4), a mitochondria stress transcription factor that is mediated by the integrated stress response pathway, is activated in response to mitochondria stress induced by Au-Macl (FIG. 26G)

[0240] Example 49: Mitofusin degradation induced by Au-Macl is mediated by PINK1 and PARKIN and requires the Ubiquitin proteasome system.

[0241] Mitochondria dysfunction induced by perturbation of mitochondria processes can trigger selective clearance of damaged mitochondria, a process known as mitophagy. This process is known to be mediated by the PINK1 / P ARKIN pathway. In the event of mitophagy, PINK1 accumulates on damaged mitochondria and serves as a signal to allow for the selective translocation of PARKIN from the cytosol to the damaged mitochondria to facilitate clearance. Given the membrane depolarization effect of Au-Macl on the ovarian cancer cells and the consequent decrease in expression of fusion proteins, the expression levels of PINK 1 and PARKIN was examined in UWB 1.289 and UWB1.289-P1atR cells treated with l uM Au-Macl . A decrease in the expression levels of total Parkin was observed, consistent with Parkin translocation from the cytosol to the mitochondria within 24 h of exposure to Au-Macl (FIG. 27A). Also, 24 h exposure of the cells to Au-Macl caused an increase in the expression level of Pink 1 as examined by immunobloting (FIG. 27A).

[0242] Recent studies have shown that Parkin (an E3-ubiquitin ligase), upon translocation to damaged mitochondria, ubiquitinates mitofusins, causing their degradation in a proteasome dependent manner. Incubation of UWB1.289 and UWB1.289-PlatR cells with the known proteasome inhibitor. MG132, inhibited Au-Macl induced decrease in MFN1 expression level (FIG. 27B). MFN1 ubiquitination was further confirmed by immunoprecipitating MFN1 from control and Au-Macl treated UWB 1 289 and UWB 1.289-PiatR cells (FIG. 27B). The immunoprecipitated MFNl samples were subjected to western blotting and the blots were probed with ubiquitin antibody; ubiquitinated MFNl molecular weight was detected (FIG. 27C, 27D) This suggests that Au-Macl induced depletion of MFNl is via the ubiquitin-proteasome system (UPS). Further confirmation of mitophagy induction due to Au-Macl mitochondria perturbations was seen via immunoblotting of phospho-ubi quitin (Ser65) in Au-Macl treated UWB1.289 and UWB1.289-PlatR ceils (FIG. 27E). PINK1 is recognized as the only identified Ser65 -ubiquitin kinase. Hence, phosphorylation of ubiquitin at Ser65 by PINK 1 due to mitochondria stress is a key marker of mitophagy induction.

[0243] Example 50: Au-Macl inhibits OC tumor growth in vivo in OC xenograft model.

[0244] Having established the efficacy of Au-Macl on ovarian cancer cell growth in vitro, efficacy was examined in vivo. The in vivo effect of Au-Macl on tumor growth was examined in SKOV-3 xenograft tumor mouse model (FIG. 28A, 28B). Either vehicle or Au- Macl was administered via intraperitoneal (IP) injection at 5mg / kg dose. Analyses of tumor volume show that Au-Macl effectively suppressed tumor growth while mice body weight was unaffected (FIG. 28A, 28B). Additionally, serum chemistry tests were performed to further evaluate the safety profile of Au-Macl, no significant changes were observed (data not shown). The data suggest that Au-Macl is a potent gold-based molecule with utility as an anticancer agent for treatment of ovarian cancer.

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[0246] It will be understood that various details of the presently disclosed subject matter can be changed without departing from the scope of the subject matter disclosed herein. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.

Claims

CLAIMSWhat is claimed is:

1. A compound of the formula:whereinW is selected from the group consisting of , an amino acid, a polypeptide, a nucleotide, and a polyethylene glycol (PEG);Z is selected from the group consisting of covalent bond, -NH-alkyl-(C=O)-, -(O)n- where n is 1-8, and -(CH2)n- where n is 1-8; each R is independently selected from the group consisting of: H, -NO2, -NH2, -NR1^ -OH, -OR1, -O(C=O)R1, -NH(C=O)R1, -CH3, -(CH2)nCH3where n is 1-9, Rx, -(C=O)H, -Rxis F, Br, Cl, or I;R1is - (CH2)n-CH3where n=l-8;R2is O or a functional moiety; andQ is a pharmaceutically-acceptable anion.

2. The compound of claim 1, of the formula:Z is selected from the group consisting of covalent bond, -NH-alkyl-(C=O)-, -(O)n- where n is 1-8, and -(CH2)n- where n is 1-8; each R is independently selected from the group consisting of: H, -NO2, -NH2, -NR1!, -OH, -OR1, -O(C=O)R1, -NH(C=O)R1, -CH3, -(CH2)nCH3where n is 1-9, Rx, -(C=O)H, -Rxis F, Br, Cl, or I;R1is - (CH2)n-CH3where n=l-8;R2is O or a functional moiety; andQ is a pharmaceutically-acceptable anion.

3. The compound of claim 1 or 2, wherein Y is selected from the group consisting ofwherein each R is independently selected from the group consisting of: H, -NO2, -NH2, - NR’2, -OH, -OR1, -O(C=O)R1, -NH(C=O)R1, -CH3, -(CH2)nCH3where n is 1-9, Rx, -(C=O)H, -Rxis F, Br, Cl, or I;R1is - (CH2)n-CH3where n=l-8; andR2is O or a functional moiety.

4. The compound of claim 1, of the formula:Rxis F, Br, Cl, or I;R1is - (CH2)n-CH3where n=l-8;R2is O or a functional moiety; andQ is a pharmaceutically-acceptable anion.

5. The compound of claim 1, selected from the group consisting of:each R is independently selected from the group consisting of: H, -NO2, -NH2, -NR1?, - OH, -OR1, -O(C-O)R', -NH(C~O)R', -CH3, -(CH2)nCH3where n is 1-9, Rx, -(C=O)H, -Rxis F, Br, Cl, or I;R1is - (CH2)n-CH3where n=l-8;R2is O or a functional moiety; andQ is a pharmaceutically-acceptable anion.

6. The compound of claim 1, of the formula:Q is a pharmaceutically-acceptable anion.

7. The compound of claim 1, selected from the group consisting of:

8. The compound of claim 1, of the formulawherein X is selected from the group consisting<img src='' class="img-anchor img-center" img-id="IMGF000092_0001" / >9. The compound of claim 1, of the formula:Y is alkyl or aryl;Z is selected from the group consisting of covalent bond, -NH-alkyl-(C=O)-, -(O)n- where n is 1-8, and -(CH2)n- where n is 1-8; each R is independently selected from the group consisting of: H, -NO2, -NH2, -NR1!, -OH, -OR1, -O(C=O)R1, -NH(C=O)R1, -CH3, -(CH2)nCH3where n is 1-9, Rx, -(C=O)H, -Rxis F, Br, Cl, or I;R1is - (CH2)n-CH3where n=l-8; andR2is O or a functional moiety; andQ is a pharmaceutically-acceptable anion.

10. The compound of claim 9, of the formula:wherein Q is a pharmaceutically-acceptable anion.

11. The compound of claim 1, selected from the group consisting ofwherein Y is alkyl or aryl;Z is selected from the group consisting of covalent bond, -NH-alkyl-(C=O)-, -(O)nwhere n is 1-8, and -(CH2)n- where n is 1-8;each R is independently selected from the group consisting of: H, -NO2, -NH2, -NR1?, -OH, -OR1, -O(C-O)R', -NH(C~O)R', -CH3, -(CH2)nCH3where n is 1-9, Rx, -(C=O)H, -Rxis F, Br, Cl, or I;R1is - (CH2)n-CH3 where n=l-8;R2is O or a functional moiety; andQ is a pharmaceutically-acceptable anion.

12. The compound of claim 1, of the formula:wherein Y is selected from the group consisting ofpharmaceutically-acceptable anion.

13. The compound of claim 1, of the formula:wherein Z is selected from the group consisting of covalent bond, -NH-alkyl-(C=O)-, -(O)n- where n is 1-8, and -(CH2)n- where n is 1-8; and Q is a pharmaceutically-acceptable anion.

14. The compound of claim 1, selected from the group consisting of:Rxis F, Br, Cl, or I;R1is - (CH2)n-CH3where n=l-8; andQ is a pharmaceutically-acceptable anion.

15. The compound of claim 1, of the formula:wherein Q is a pharmaceutically-acceptable anion.

16. The compound of claim 1, of the formula:wherein Q is a pharmaceutically-acceptable anion.

17. The compound of claim 1, of the formula:wherein X is selected from the group consisting<img src='' class="img-anchor img-center" img-id="IMGF000099_0002" / >R2is O or a functional moiety; and Q is a pharmaceutically-acceptable anion.

18. The compound of claim 17, of the formula:wherein Q is a pharmaceutically-acceptable anion.

19. The compound of any one of claims 8, 9, 11, and 17, wherein Y is selected from theRxis F, Br, Cl, or I;R1is - (CH2)n-CH3 where n=l-8; andR2is O or a functional moiety.

20. A pharmaceutical composition comprising the compound of any one of claims 1-19 and a pharmaceutically-acceptable carrier.

21. A method of conferring anti-cancer activity to a cancer cell, comprising: contacting a cancer cell with an effective amount of the compound of any one of claims 1-19.

22. The method of claim 21, wherein the conferring anti-cancer activity results in one or more of inhibiting proliferation of the cancer cell, inhibiting metastasis, and killing the cancer cell.

23. The method of claim 21, wherein the cell is a cultured cell.

24. The method of claim 21, wherein the cell is in a subject.

25. The method of claim 24, wherein the subject is a mammal.

26. The method of claim 21, wherein the cancer is a breast cancer.

27. The method of claim 26, wherein the breast cancer is triple negative breast cancer.

28. The method of claim 21, wherein the cancer is ovarian cancer.

29. The method of claim 21, wherein the cancer is lung cancer.

30. The method of claim 21, wherein the cancer is a glioblastoma.

31. The method of claim 21 , wherein the cancer is a platinum resistant cancer.

32. The method of claim 31, wherein the cancer is a platinum resistant gynecologic cancer.

33. The method of claim 32, wherein the cancer is a platinum resistant ovarian tumor.

34. A method of modulating mitochondrial function in a cell, comprising: contacting a cell with an effective amount of the compound of any one of claims 1-19.

35. The method of claim 34, wherein the cell is a cancer cell.

36. The method of claim 34, wherein the cell is a cultured cell.

37. The method of claim 34, wherein the cell is in a subject.

38. The method of claim 34, wherein the subject is a mammal.

39. A method of inhibiting oxidative phosphorylation (OXPHOS) in a cell, comprising: contacting a cell with an effective amount of the compound of any one of claims 1-19.

40. A method of reducing viability of cancer stem cells (CSCs), comprising: contacting a cell with an effective amount of the compound of any one of claims 1-19.

41. A method of targeting HM0X2 in a cell, comprising: contacting a cell with an effective amount of the compound of any one of claims 1-19.

42. Use of the compound of any one of claims 1-19 in a medicament for the treatment of a cancer.'43. Use of the compound of any one of claims 1-19 in a medicament for the treatment of a condition involving mitochondrial dysfunction.

44. Use of the compound of any one of claims 1-19 in a medicament for inhibiting oxidative phosphorylation (OXPHOS).

45. Use of the compound of any one of claims 1-19 in a medicament for reducing viability of cancer stem cells (CSCs).

46. Use of the compound of any one of claims 1-19 in a medicament for targeting HM0X2 in a cell.

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