FIELD

[0001] This disclosure relates to tri -phenyl -phosphonium (TPP) derivative compounds, or

TPP-derivatives, having an improved selectivity for mitochondria to target bulk cancer cells, cancer stem cells, and normal senescent cells.

BACKGROUND

[0002] Conventional cancer therapies, such as irradiation, alkylating agents, and anti- metabolites, work by selectively eradicating fast-growing cancer cells through interfering with cell growth and DNA replication mechanisms. Tumors often recur after such therapies, indicating that not all cancer cells were eradicated. Cancer stem cells (CSCs) are tumor-initiating cells (TICs) that appear to be the biological basis of treatment failure, tumor recurrence and distant metastasis, ultimately leading to poor clinical outcome in cancer patients. As a consequence, new therapies are urgently needed, to specifically target and eradicate CSCs.

[0003] Interestingly, recent studies indicate that one unique feature of CSCs is a characteristic increase in mitochondrial mass, which may reflect a more strict dependence on mitochondrial function or OXPHOS. Several independent lines of evidence support the idea that increased mitochondrial biogenesis or higher levels of mitochondrial protein translation may occur in CSCs. For example, unbiased proteomics analysis directly shows that mitochondrial mass is elevated in CSCs.

[0004] Moreover, MitoTracker (a mitochondrial fluorescent dye) can be used successfully as a marker to identify and purify CSCs. More specifically, the “Mito-high” cell population shows the greatest capacity for i) anchorage-independent growth and ii) tumor-initiating ability in vivo. [0005] High telomerase activity also directly correlates with high mitochondrial mass and the ability of CSCs to undergo proliferative expansion. Similarly, high mitochondrial mass in CSCs was also specifically associated with mitochondrial reactive oxidative species (ROS) production (hydrogen peroxide) and could be targeted with either: i) mitochondrial anti-oxidants, ii) inhibitors of mitochondrial biogenesis (doxycycline) or OXPHOS, and even iii) inhibitors of cell proliferation (palbociclib, a CDK4/6 inhibitor).

[0006] There exists a need in the art for novel and effective anti-cancer therapies, including the development of not only new anti-cancer compounds, but also methods for identifying new classes of compounds having anti-cancer efficacy. Ideal compounds are selective towards cancer cells, including TICs, yet non-toxic to normal cells. This also includes compounds and moieties that specifically target bulk cancer cells, cancer stem cells, and normal senescent cells, for targeted delivery of therapeutic agents.

SUMMARY

[0007] This disclosure describes a new approach for the eradication of CSCs and related therapies, through the use of novel mitochondrial inhibitors conjugated with a fatty acid targeting signal. Disclosed herein are new strategies for identifying novel and non-toxic mitochondrial targeting signals, including the identity of specific compounds that may be used to enhance the anti-mitochondrial effects of other therapeutic agents. Disclosed herein are demonstrative embodiments of TPP-conjugate compounds, referred to as TPP-derivatives. The mitochondrial uptake of a TPP-derivative may be further increased through the substitution of one or more fatty acid targeting signals, as described herein.

[0008] Tri-phenyl-phosphonium (TPP) serves as a chemical mitochondrial targeting signal, and also represents a new avenue for safe and effective anti-cancer therapies. Described herein are TPP-derivative compounds that have been developed having a strong preference for uptake in cancer cells (e.g., bulk cancer cells, cancer stem cells, and energetic cancer stem cells), as well as normal but senescent cells. Importantly, TPP-derivatives described herein are non-toxic in healthy cells and normal fibroblasts, but potently target CSC propagation, with an IC-50 as low as 500 nM. As disclosed herein, TPP-derivative 2-butene- 1 ,4-bis-TPP is an example of an effective TPP compound for targeting CSC propagation, among other potential therapies. Conjugating this TPP-derivative with a fatty acid targeting signal further improves the selectivity and activity. [0009] The present approach may be used to treat and/or prevent tumor recurrence, metastasis, drug resistance, and/or radiotherapy resistance. Anti-cancer treatments often fail because the tumor recurs or metastasizes, particularly after surgery. Also, drug resistance and radiotherapy resistance are common reasons for cancer treatment failure. It is believed that CSC mitochondrial activity may be, at least in part, responsible for these causes of treatment failure. Embodiments of the present approach may be used in situations where conventional cancer therapies fail, and/or in conjunction with anti-cancer treatments to prevent failure due to tumor recurrence, metastasis, drug resistance, and/or radiotherapy resistance. The present approach may also take the form of a method for targeting a therapeutic agent to a cancer stem cell mitochondria, by chemically modifying the therapeutic agent with at least one TPP-derivative compound.

[0010] As used herein, a TPP-derivative is an organic chemical compound derived from, or including, TPP. For example, a TPP-derivative compound may be 2-butene- 1,4-bis-TPP; 2- chlorobenzyl-TPP; 4-chlorobenzyl-TPP; 3-methylbenzyl-TPP; 2,4-dichlorobenzyl-TPP; 1- naphthylmethyl-TPP; p-xylylenebis-TPP; dodecyl-TPP; and derivatives of any of the foregoing. It should be appreciated that the foregoing list is not an exhaustive list of TPP-derivatives that inhibit CSC propagation. As will be appreciated, however, the conjugated moiety(ies) can have a significant impact on whether the TPP-derivative has anti-cancer or other beneficial properties, as well as the potency of those properties. In particular, a TPP-derivative may be conjugated with a fatty acid targeting signal to increase the mitochondrial uptake, and hence the efficacy, of the TPP- derivative.

[0011] The present approach may take the form of a method for treating cancer, in which a pharmaceutically effective amount of at least one TPP-derivative compound is administered. The present approach may also take the form of methods and pharmaceutical compositions for treating and/or preventing tumor recurrence, metastasis, drug resistance, and/or radiotherapy resistance, in which a pharmaceutically effective amount of at least one TPP-derivative compound is administered, either in conjunction with or after cancer therapy. The TPP-derivative compound may be administered with a mitochondrial inhibitor or other therapeutic agent, thereby increasing the agent’s uptake in cancer cells with little or no effect on normal, healthy cells. The TPP- derivative compound may be 2-butene- 1,4-bis-TPP; 2-chlorobenzyl-TPP; 3-methylbenzyl-TPP; 2,4-dichlorobenzyl-TPP; 1-naphthylmethyl-TPP; p-xylylenebis-TPP, or dodecyl-TPP. In some embodiments, the TPP-derivative is conjugated with a fatty acid targeting signal. In preferred embodiments, the fatty acid targeting signal has a linear, saturated aryl chain. In some embodiments, there may be more than one TPP-derivative. Some embodiments of the present approach may take the form of a use of a TPP-derivative as disclosed herein, in the manufacture of a medicament for treating cancer, preventing or reducing the likelihood of tumor metastasis and/or recurrence, and/or increasing the effectiveness of another cancer treatment (e.g., another chemotherapeutic, radiotherapy, phototherapy, etc.).

[0012] It should be appreciated that the TPP-derivative compound selectively targets cancer stem cells in embodiments of the present approach. Further, in some embodiments, the at least one TPP-derivative compound selectively targets normal senescent cells. TPP-derivative compounds may be minimally toxic and, in some embodiments, non-toxic, to normal healthy cells. [0013] The present approach may take the form of a composition having, as an active ingredient, at least one TPP-derivative compound. The ingredient may be active towards targeting CSCs and inhibiting CSC propagation. For example, the pharmaceutical composition may be an anti-cancer pharmaceutical composition having, as its active ingredient, at least one TPP- derivative compound. In some embodiments, for example, the active ingredient is 2 -butene- 1,4- bis-TPP. As another example, the active ingredient in some embodiments is a derivative of 2- butene-l,4-bis-TPP having a fatty acid moiety, such as myristate.

[0014] Embodiments of the pharmaceutical composition may eradicate bulk cancer cells, cancer stem cells, and normal senescent cells. As disclosed herein, the TPP-compounds described are highly selective towards cancer cells and, more specifically, cancer stem cells and tumor initiating cells having a higher energetic state. Further, TPP-derivative compounds of the present approach are generally non-toxic towards normal healthy cells.

[0015] TPP-derivatives may also eradicate senescent cells, thereby reducing and/or eliminating various aging-related diseases. The present approach may therefore take the form of a method for treating an affliction through administering a pharmaceutically effective amount of at least one TPP-derivative compound. The TPP-derivative compound may be administered with one or more additional therapeutic agents, such as agents having anti-mitochondrial effects. The affliction may be, for example, cancer, an age-associated illness, senescence-associated secretory phenotype, or the effects of aging, such as atherosclerosis, cardiovascular disease, cancer, arthritis, cataracts, osteoporosis, type 2 diabetes, obesity, metabolic syndrome, hypertension, Alzheimer's disease, chronic inflammation, neuro-degeneration, muscle-wasting (sarcopenia), loss of skin elasticity, greying of the hair, male-pattern baldness, age spots, skin imperfections, and keratosis.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figures 1A and IB show the effect of TPP Compound 1 (2-butene- l,4-bis-TPP)on cell viability and intracellular ATP levels in MCF-7 human breast cancer cells.

[0017] Figures 2A and 2B illustrate the effect of TPP-derivatives on cell viability and intracellular ATP levels in MCF-7 human breast cancer cells, for TPP Compounds 2 (2- chlorobenzyl-TPP) and 3 (3-methylbenzyl-TPP).

[0018] Figures 3 A and 3B show the effect of TPP-derivatives on cell viability and intracellular ATP levels in MCF-7 human breast cancer cells for TPP Compounds 4 (2,4- dichlorobenzyl-TPP) and 5 (1-naphthylmethyl-TPP).

[0019] Figure 4 illustrates the effects of TPP-derivatives on cell viability and intracellular

ATP levels in normal fibroblasts (hTERT-BJ 1) and human breast cancer cells (MCF-7).

[0020] Figure 5 illustrates impaired mitochondrial function of MCF-7 cells after treatment with TPP Compound 1 (2-butene- 1,4-bis-TPP).

[0021] Figures 6A and 6B illustrate impaired mitochondrial function of MCF-7 cells after treatment with TPP Compounds 2 (2-chlorobenzyl-TPP) and 3 (3-methylbenzyl-TPP).

[0022] Figures 7A and 7B show impaired mitochondrial function of MCF-7 cells after treatment with TPP Compounds 4 (2,4-dichlorobenzyl-TPP) and 5 (1-napthylmethyl-TPP).

[0023] Figure 8 illustrates the differential inhibition of the mammosphere-forming activity of MCF-7 breast CSCs, after treatment with TPP-derivatives. [0024] Figure 9 shows a demonstrative approach for identifying mitochondrial inhibitors to target CSC propagation according to the present approach.

[0025] Figure 10 shows the relative quantity of metastasis using the CAM assay for an embodiment of the present approach.

DESCRIPTION

[0026] The following description illustrates embodiments of the present approach in sufficient detail to enable practice of the present approach. Although the present approach is described with reference to these specific embodiments, it should be appreciated that the present approach may be embodied in different forms, and this description should not be construed as limiting any appended claims to the specific embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the present approach to those skilled in the art.

[0027] This description uses various terms that should be understood by those of an ordinary level of skill in the art. The following clarifications are made for the avoidance of doubt. The terms “treat,” “treated,” “treating,” and “treatment” include the diminishment or alleviation of at least one symptom associated or caused by the state, disorder or disease being treated, in particular, cancer. In certain embodiments, the treatment comprises diminishing and/or alleviating at least one symptom associated with or caused by the cancer being treated, by the compound of the invention. For example, treatment can be diminishment of one or several symptoms of a cancer, or complete eradication of a cancer. In some embodiments, the treatment comprises causing the death of a category of cells, such as CSCs, of a particular cancer in a host, and may be accomplished through preventing cancer cells from further propagation, and/or inhibiting CSC function through, for example, depriving such cells of mechanisms for generating energy.

[0028] The terms “cancer stem cell” and “CSC” refer to the subpopulation of cancer cells within tumors that have capabilities of self-renewal, differentiation, and tumorigenicity when transplanted into an animal host. Compared to “bulk” cancer cells, CSCs have increased mitochondrial mass, enhanced mitochondrial biogenesis, and higher activation of mitochondrial protein translation. As used herein, a “circulating tumor cell” is a cancer cell that has shed into the vasculature or lymphatics from a primary tumor and is carried around the body in the blood circulation. The CellSearch Circulating Tumor Cell Test may be used to detect circulating tumor cells.

[0029] Under the present approach, a TPP-derivative compound may be conjugated with a fatty acid targeting signal to improve the uptake and, as a result, the potency, of the TPP- derivative with respect to targeting and eradicating CSCs, and in particular, preventing or reducing the likelihood of metastasis. The substituted TPP-derivative compounds of the present approach have the general formula X — P ⁺ — (Ph) ₃ — Y, wherein:

X represents an organic moiety, and may be selected from alkanes, cyclic alkanes, alkane -based derivatives, alkenes, cyclic alkenes, alkene-based derivatives, alkynes, alkyne -based derivative, ketones, ketone-based derivatives, aldehydes, aldehyde -based derivatives, carboxylic acids, carboxyls, carboxylic acid-based derivatives, ethers, ether- based derivatives, esters and ester-based derivatives, amines, amino-based derivatives, amides, amide-based derivatives, monocyclic or polycyclic arene, heteroarenes, arene- based derivatives, heteroarene- based derivatives, phenols, phenol-based derivatives, benzoic acid, benzoic acid-based derivatives, and a TPP-derivative, any of which may be substituted or unsubstituted;

P+ is quaternary phosphorus cation;

Ph is substituted or unsubstituted phenyl; and Y is a fatty acid targeting signal, as described herein.

[0030] Embodiments of the present approach may take the form of pharmaceutical compositions. Compounds of the present approach, when used in a pharmaceutical composition, may be administered by any route appropriate, including, but not limited to, oral, parenteral, transdermal, topical, nasal, aerosol, intrapulmonary, etc. It should be appreciated that the pharmaceutical compound(s) of the present approach may be administered to the subject through any suitable approach, as would be known to those having an ordinary level of skill in the art. [0031] It should also be appreciated that the amount of compound and the timing of its administration in a pharmaceutical composition may depend on the individual subject being treated (e.g., the age and body mass, among other factors), on the manner of administration, on the pharmacokinetic properties of the particular active compound(s), and on the judgment of the prescribing physician. Thus, because of subject to subject variability, any dosages described herein are intended to be initial guidelines, and the physician can titrate doses of the compound to achieve the treatment that the physician considers appropriate for the subject. In considering the degree of treatment desired, the physician can balance a variety of factors such as age and weight of the subject, presence of preexisting disease, as well as presence of other diseases. The dosage of a pharmaceutical composition will thus vary, depending on factors such as the form of administration, as well as the age, weight, physical condition, and diagnosis of the subject to be treated. In some instances, a treatment may commence using a small dose, and the dose may be increased until a desired effect is achieved. In some embodiments, the dose may be a pharmaceutically effective amount of the compound. The phrase “pharmaceutically effective amount,” as used herein, indicates an amount necessary to administer to a host, or to a cell, tissue, or organ of a host, to achieve a therapeutic result, such as regulating, modulating, or inhibiting protein kinase activity, e.g., inhibition of the activity of a protein kinase, or treatment of cancer. For example, the dosage of a pharmaceutical composition in some embodiments may be about 1 mg/kg up to about 100 mg/kg body weight of the subject receiving treatment in some embodiments, e.g., about 2 mg/kg to about 75 mg/kg body weight of the subject, e.g., about 5 mg/kg to about 50 mg/kg body weight of the subject, e.g., about 10 mg/kg to about 50 mg/kg body weight of the subject. Higher or lower doses are contemplated and are, therefore, within the scope of the present approach. The dose may also vary during the course of treatment, and may precede, or follow, other cancer treatments, including chemotherapy, radiotherapy, and phototherapy. In some embodiments, the dose will depend on whether the subject is in a fasted state, e.g., has not consumed calories for a period, e.g., 8 hours, 12 hours, 16 hours, 24 hours, and so on. The efficacy of some embodiments, and for some treatments, may be improved when the subject is in a fasted state. A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0032] As used herein, the term “derivative” is a chemical moiety derived or synthesized from a referenced chemical moiety. As used herein, a conjugate is a compound formed by the joining of two or more chemical compounds. For example, a conjugate of a TPP-derivative and a fatty acid results in a compound having a TPP-derivative moiety and a moiety derived from the fatty acid As used herein, a fatty acid is a carboxylic acid with an aliphatic chain, which is either saturated or unsaturated. Preferred embodiments have a fatty acid moiety with a linear and saturated aliphatic chain with between (and including) 1 and 50 carbons, and more preferably between (and including) 5 and 25 carbons, and more preferably, between (and including) 10 and 20 carbons. Examples of fatty acids include short chain fatty acids (i.e., having 5 or fewer carbon atoms in the chemical structure), medium-chain fatty acids (i.e., having 6-12 carbon atoms in the chemical structure), and long chain fatty acids (i.e., having 13-21 carbon atoms in the chemical structure). Examples of saturated fatty acids include lauric acid (CH ₃ (CH ₂ ) ₁₀ COOH) , palmitic acid (CH ₃ (CH ₂ ) ₁₄ COOH), stearic acid (CH ₃ (CH ₂ ) ₁₆ COOH), and myristic acid (CH ₃ (CH ₂ ) ₁₂ COOH). Oleic acid (CH ₃ (CH ₂ ) ₇ C H C H (CH ₂ ) ₇ COO H ) is an example of a naturally occurring unsaturated fatty acid. References may also be made to the salt or ester of a fatty acid, as well as its fatty amide moiety. For example, myristic acid may be referred to as myristate, and oleic acid may be referred to as oleate. A fatty acid moiety may also be a carboacyl of the fatty acid, i.e., a group formed by the loss of a hydroxide group of a carboxylic acid. In some embodiments, a fatty acid moiety may be bonded to a therapeutic agent through an amide bond. As an example, a myristic acid conjugate may have a fatty acid moiety (CH ₃ (CH ₂ ) ₁₂ CO-NH-, where the tertiary nitrogen is bonded to the therapeutic agent: and n is an integer from 0 to 49, and preferably is 4 to 24, and preferably is 9 to 19. This may result when the myristate moiety is conjugated through myristoylation, resulting in a tetradecanarnide (or myristamide) group. For simplicity, embodiments having a carboacyl are nonetheless referred to as having a fatty acid moiety.

[0033] The inventors previously identified the approach of inhibiting mitochondrial function in CSCs and TICs as an avenue for eradicating cancer cells. Given the role of mitochondrial biogenesis in tumor proliferation, the inventors recognized that mitochondrial targeting represents a valuable characteristic for anti-cancer therapies. Tri-phenyl-phosphonium (TPP) is a well-established chemical mitochondrial targeting signal. Cargo molecules covalently attached to TPP accumulate within the mitochondria of living cells. However, successful anti- cancer therapies require targeting cancer cells, as opposed to normal cells. As discussed herein, certain TPP-derivatives have been developed that not only selectively target cancer cell mitochondria, but also have minimal-to-no side-effects in normal cells.

[0034] In order to identify new molecules that can be used to target mitochondria within

CSCs, the inventors screened a variety of TPP-derivatives as described herein, by employing CellTiter-Glo to measure intracellular levels of ATP in adherent cancer cells (MCF-7) in 96-well plates. As 85% of cellular ATP is normally derived from mitochondrial metabolism, ATP levels are an excellent read-out to monitor mitochondrial function. In parallel, the same 96-well plates were also stained with Hoechst 33342, to measure DNA content, to gauge cell viability. Therefore, the inventors selected 9 TPP derivatives and subjected them to screening in our assay system. The chemical structures of these TPP-derivatives are shown below.

2-butene- 1 ,4-bis-TPP

(Compound 1)

2-chlorobenzyl-TPP

(Compound 2) 3 -methylbenzyl-TPP

(Compound 3)

2,4-dichlorobenzyl-TPP

(Compound 4)

1 -naphthylmethyl-TPP ((Compound 5) mito-TEMPO

(2-(2,2,6,6-Tetramethylpiperidin-l-oxyl-4- ylamino)-2-oxoethyl)triphenylphosphonium chloride)

(Compound 6) cyanomethyl-TPP (Compound 7) p-xylylene-bis-TPP

(Compound 8)

4-cyanobenzyl-TPP (Compound 9)

[0035] TPP, as a mitochondrial targeting signaling, is non-toxic in normal cells. The inventors recognized that TPP-derivative compounds could be developed to inhibit mitochondrial function in CSCs, and developed the approach shown in Figure 9 for identifying such compounds. To demonstrate this approach, the inventors used an ATP depletion assay to screen the activity of the nine TPP-derivatives identified above.

[0036] Five of the screened TPP -related compounds significantly suppressed ATP levels, which yields a hit-rate of more than 50%. All five positive hit compounds were subjected to functional validation with the Seahorse XFe96, to quantitate their effects on the mitochondrial oxygen consumption rate (OCR). Remarkably, these TPP hit compounds were non-toxic in normal human fibroblasts and did not affect their viability or ATP production, showing striking selectivity for cancer cells. Most importantly, these top hit compounds successfully blocked CSC propagation, as shown by employing the 3D spheroid assay. For example, 2-butene- 1,4-bis-TPP was that most potent molecule identified in the tested TPP-derivatives identified above, which targeted CSC propagation with an IC-50 < 500 nM. Interestingly, 2-butene- 1,4-bis-TPP contains two TPP groups. This suggests that the use of a bis-TPP moiety may function as a “dimeric” or “polymeric” signal for the more effective targeting of mitochondria in CSCs. Further studies are contemplated to continue exploring the potential of bis-TPP as a highly effective therapeutic agent. [0037] Notably, five out of the nine TPP-derivative compounds were “positive hits” that significantly reduced ATP levels. These positive hits included: 2 -butene- 1,4-bis-TPP, 2- chlorobenzyl-TPP, 3-methylbenzyl-TPP, 2,4-dichlorobenzyl-TPP and 1-naphthylmethyl-TPP. This represents a hit rate of >50% of the TPP-derivatives subject to the demonstrative study. However, two compounds were completely ineffective in reducing ATP levels (see Table 1, below). This finding is consistent with previous studies showing that the TPP moiety is not intrinsically toxic for normal cell mitochondria.

[0038] After initial screening, the five positive hit compounds were then subjected to further validation studies, shown in Figures 1-3, demonstrating that these TPP compounds are highly active in the range of 0.5 to 2 mM. Based on this initial analysis, 2-butene- 1,4-bis-TPP demonstrated the highest potency of the positive hit TPP-derivatives.

[0039] Figure 1A shows the effect of TPP Compound 1, 2-butene- 1,4-bis-TPP, on cell viability and intracellular ATP levels in MCF-7 human breast cancer cells. Cell viability and intracellular ATP levels were determined in the same treated samples. Hoechst staining (%) (shown in black bars); ATP levels (%) indicated in the white bars. MCF-7 cells were treated for 72h. Data are represented as mean +/- SEM. Note that 2-butene- 1,4-bis-TPP depletes ATP levels, relative to cell number. **p < 0.01; ***p < 0.001; indicates significance, all relative to the control. Figure IB shows a magnified image of control and treated cell plates.

[0040] Figures 2A and 2B show the effect of TPP Compound 2 (2-chlorobenzyl-TPP) and

TPP Compound 3 (3-methylbenzyl-TPP) on cell viability and intracellular ATP levels in MCF-7 human breast cancer cells. Cell viability and intracellular ATP levels were determined in the same treated samples. Hoechst staining (%) (shown in black bars); ATP levels (%) indicated in white bars. MCF-7 cells were treated for 72h. Data are represented as mean +/- SEM. Note that both 2- chlorobenzyl-TPP and 3-methylbenzyl-TPP progressively deplete cellular ATP levels. *p < 0.05; **p < 0.01; ***p < 0.001; indicates significance, all relative to the control.

[0041] Figures 3 A and 3B show the effect of TPP Compound 4 (2,4-dichlorobenzyl-TPP) and TPP Compound 5 (1-naphthylmethyl-TPP) on cell viability and intracellular ATP levels in MCF-7 human breast cancer cells. Cell viability and intracellular ATP levels were determined in the same treated samples. Hoechst staining (%) (shown in black bars); ATP levels (%) indicated in white bars. MCF-7 cells were treated for 72h. Data are represented as mean +/- SEM. Note that both 2,4-dichlorobenzyl-TPP and 1-naphtylmethyl-TPP progressively deplete cellular ATP levels. *p < 0.05; **p < 0.01; ***p < 0.001; indicates significance, all relative to the control.

[0042] Figure 4 shows the effects of TPP-derivatives on cell viability and intracellular ATP levels in normal fibroblasts (hTERT-BJ1) and human breast cancer cells (MCF-7). Cell viability and intracellular ATP levels were determined in the same treated samples. Hoechst staining (%) of hTERT-BJ1 human fibroblasts (black); ATP level (%) of hTERT-BJ1 human fibroblasts (dotted); Hoechst staining (%) of MCF-7 cells (inclined lines); ATP level (%) of of MCF-7 cells (white). TPP treatments at 1 mM, 72h. Data are represented as mean +/- SEM. *p < 0.05; **p < 0.01; indicates significance, all relative to the control.

[0043] As can be seen in Figure 4, these TPP-derivatives are relatively non-toxic in normal human fibroblasts (hTERT-BJ1), but are preferentially active in cancer cells (MCF-7). For example, in human fibroblasts, 2-butene- 1 ,4-bis-TPP had no effect on cell viability and only mildly reduced ATP levels by 25%. In contrast, at the same concentration ( 1 mM) in MCF-7 cancer cells, 2-butene- 1,4-bis-TPP reduced cell viability by nearly 65% and decreased ATP levels by almost 85%. Therefore, 2-butene- 1,4-bis-TPP was 2.8-fold more effective at reducing cell viability in cancer cells (versus fibroblasts). Similarly, 2-butene- 1,4-bis-TPP was 4.7-fold more effective at reducing ATP levels in cancer cells, relative to normal fibroblasts.

[0044] Embodiments of the present approach relate to identifying TPP-derivatives compounds that target mitochondria in CSCs and represent potential anti-cancer therapies. To further validate that the ATP level reduction of the TPP compounds was indeed due to the inhibition of mitochondrial function, mitochondrial oxygen consumption rates (OCR) were directly measured using the Seahorse XFe96 metabolic flux analyser. The results are shown in Figures 5-7B. All five TPP Compounds behaved similarly, and effectively reduced basal mitochondrial respiration, with an IC-50 of approximately 1 mM. The identified TPP-derivatives also showed significant reduction in ATP-link respiration, maximal respiration, and spare respiratory capacity.

[0045] Figure 5 illustrates the impaired mitochondrial function of MCF-7 cells after treatment with TPP Compound 1. Figures 6A and 6B illustrate the impaired mitochondrial function of MCF-7 cells after treatment with either TPP Compound 2 or TPP Compound 3. Figures 7A and 7B show the impaired mitochondrial function of MCF-7 cells after treatment with TPP Compound 4 and TPP Compound 5, respectively. Oxygen consumption rate (OCR) was measured with a Seahorse XF96 Extracellular Flux Analyzer. Data are represented as mean +/- SEM. Note that 2- butene- 1,4-bis-TPP effectively inhibits mitochondrial oxygen consumption. For Fig. 5, **p <0.01; ***p < 0.001; indicates significance, all relative to the control. Note that 2-chlorobenzyl-TPP and 3-methylbenzyl-TPP both effectively inhibit mitochondrial oxygen consumption. For Figs. 6A and 6B, **p < 0.01; ***p < 0.001; indicates significance, all relative to the control. Note that 2,4- dichlorobenzyl-TPP and 1-naphtylmethyl-TPP both effectively inhibit mitochondrial oxygen consumption. And for Figs. 7A and 7B, **p < 0.01; ***p < 0.001; indicates significance, all relative to the control.

[0046] Following validation, the inventors evaluated the effects of these TPP-derivative compounds on the propagation of CSCs, using the mammosphere assay as a read-out. Figure 8 shows the differential inhibition of the mammosphere-forming activity of MCF-7 breast CSCs, after treatment with various TPP derivatives. The data presented in Figure 8, from left to right, are TPP-derivatives in the following order: 2,4-dichlorobenzyl-TPP (black); 1-naphthylmethyl-TPP (inclined lines); 3-methylbenzyl-TPP (dotted lines); 2-chlorobenzyl-TPP (white); 2-butene-l,4- bis-TPP (horizontal lines). Cells were treated for 5 days in mammosphere media. Data are represented as mean +/- SEM. Note that 2-butene- 1,4-bis-TPP was the most effective compound for blocking CSC propagation, with an IC-50 less than 500 nM. *p < 0.05; **p < 0.01; indicates significance, all relative to the control.

[0047] TPP-derivative 2-butene- 1,4-bis-TPP was the most effective, with an IC-50 < 500 nM. In contrast, for two of the other compounds tested (2-chlorobenzyl-TPP; 3-methylbenzyl- TPP) the IC-50 was between 1 to 5 mM. Finally, 1-naphthylmethyl-TPP was the least potent, with an IC-50 > 5 mM. Therefore, the inventors concluded that 2-butene- 1,4-bis-TPP is 2- to 10-fold more potent than the other TPP compounds, for targeting CSC propagation. This is despite the fact that the identified TPP-derivatives had nearly identical behavior in reducing mitochondrial respiration and ATP production. Therefore, another intrinsic property of 2-butene- 1,4-bis-TPP allows it to better target CSCs than the other TPP Compounds explored in the inventors’ confirmatory work. [0048] Figure 9 shows an embodiment of the method according to the present approach:

Identifying mitochondrial inhibitors to target CSC propagation. First, at S1001, prospective compounds are selected from a library and subjected to ATP-depletion assays in cancer cells. As discussed above, the inventors selected TPP-related compounds as the starting point for screening, because this ensures that all the compounds tested are targeted to mitochondria. It should be appreciated by those of ordinary skill in the art that other TPP-related compounds, or other compounds containing moieties having a demonstrated or expected capacity for targeting the mitochondrial membrane, may be selected under the present approach. Compounds that reduce ATP levels are then identified S1002, and then may be functionally validated S1003 through, as an example, an analysis mitochondrial oxygen consumption rates (OCR) such as discussed above. It should be appreciated that those having ordinary skill in the art may employ alternative assays to confirm that ATP level reduction of is due to the inhibition of mitochondrial function. Following validation, the effects of identified compounds on CSC proposition S1004 may be assessed. As discussed above, one embodiment of the present approach used mammosphere assays to assess CSC propagation effects, though those having ordinary skill in the art may use alternative approaches to assess the efficacy of an identified compound for targeting CSCs. The outcome of this approach is the identification of new compounds having CSC inhibition effects. Beneficially, as applied in the confirmatory analysis described herein, the present approach demonstrates that the identified TPP-derivatives are mitochondrial inhibitors of CSCs that are non-toxic in normal human fibroblasts, thereby effectively limiting drug toxicity.

[0049] The present approach relates to substituted TPP-derivatives, such as the 9 TPP

Compounds identified above. Below is a general formula for an example of a substituted TPP- derivative, referred to as a bis-TPP compound.

This TPP-derivative is an effective mitochondrial targeting signal for eradicating cancer stem cells (CSCs). The “dimeric” structure of bis-TPP is shown in the drawing, where R represents a chemical group or moiety. For example, R may be selected from the group consisting of hydrogen, carbon, nitrogen, sulfur, oxygen, fluorine, chlorine, bromine, iodine, carboxyl, alkanes, cyclic alkanes, alkane-based derivatives, alkenes, cyclic alkenes, alkene-based derivatives, alkynes, alkyne-based derivative, ketones, ketone -based derivatives, aldehydes, aldehyde -based derivatives, carboxylic acids, carboxylic acid-based derivatives, ethers, ether-based derivatives, esters and ester-based derivatives, amines, amino-based derivatives, amides, amide -based derivatives, monocyclic or polycyclic arene, heteroarenes, arene -based derivatives, heteroarene- based derivatives, phenols, phenol-based derivatives, benzoic acid, and benzoic acid-based derivatives.

[0050] TPP-derivatives having a dimeric structure are effective for eradicating CSCs, but substitutions can impact the potency. For example, 2-butene- 1 ,4-bis-TPP and p-xylylene-bis-TPP are both dimeric. However, p-xylylene-bis-TPP is approximately 200-fold less potent than 2- butene- 1 ,4-bis-TPP, at least in the context of ATP depletion. This demonstrates that while bis-TPP compounds may be effective for eradicating CSCs, some R groups may provide significantly more (or less) potency for targeting CSCs. Table 1 shows Hoechst staining and ATP level data for certain TPP-derivative compounds. TPP-derivative compounds Mito-Tempo, cyanomethyl-TPP, p- xylylene -bis-TPP, and 4-cyanobenzyl-TPP, showed limited activity and/or undesirable levels of toxicity, and were excluded from further analysis. Those having ordinary skill in the art will appreciate the need to evaluate each potential TPP-derivative compound’s potency.

Table 1. TPP-Derivatives showing less potency in depleting ATP.

[0051] The foregoing description demonstrates that TPP-related compounds represent a novel chemical strategy for effectively targeting “bulk” cancer cells and CSCs, while minimizing off-target side-effects in normal cells. In this context, bis-TPP represents a more potent and selective form of TPP compounds, especially for targeting CSCs. Part of this potency and selectivity may also come from the reactive double bond in the central butene moiety, as p- xylylene -bis-TPP (see Table 1 above) was -200 times less effective than 2 -butene- 1,4-bis-TPP, in reducing overall ATP levels.

[0052] Further evaluation of both efficacy and toxicity was performed on human breast tumors initiated from the MDA-MB-231 cell line in chicken embryos using the CAM Model available from Inovotion (La Tranche, France). The CAM assay showed that 2-butene- 1,4-bis- TPP inhibits tumor metastasis. For example, at all concentrations tested, between 125 mM and 500 mM, 2-butene- 1,4-bis-TPP treatment led to over a 92% inhibition of tumor metastasis compared to the control. With respect to the CAM assay, fertilized eggs were incubated at 37.5 °C with 50% relative humidity for 9 days. The graft was dropped into each egg through a small hole drilled through the eggshell into the air sac, and a 1cm ² window cut in the eggshell above the CAM. This grafting process was used for each egg in a test group. Because the grafting process is an invasive surgical act, some death is expected to occur during hours after the tumor graft. For the data reported herein, at least 10 eggs per test group were successfully grafted.

[0053] The MDA-MB-231 tumor cell line was cultivated in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin. After the 9-day incubation period, cells were detached with trypsin, washed with complete medium, and suspended in a graft medium. An inoculum of 1.10 ⁶ cells was added onto the CAM of each egg, and then eggs were randomized into test groups. Tumors were detectable within one day of the graft. The negative control group was a 0.125% DMSO in PBS, and 2-butene- 1,4-bis-TPP groups for concentrations of 125 mM, 250 mM, and 500 mM, were evaluated. Each egg in a group was treated 8 times per day. After 8 days of treatments, tumor growth was quantitatively evaluated. The upper portion of the CAM (with the tumor) was removed, washed in a PBS buffer, and then directly transferred in PFA for a 48-hour fixation. Tumors were then carefully cut away from normal CAM tissue and weighed. [0054] Metastatic invasion was quantitatively evaluated. A 1 cm ² portion of the lower

CAM was collected to evaluate the number of metastatic cells in 8 samples per group (n=8). Genomic DNA was extracted from the CAM using a commercial kit, and analyzed by qPCR with specific primers for Human Alu sequences. Calculation of Cq for each sample, mean Cq and relative amounts of metastases for each group were directly managed by the Bio-Rad® CFX Maestro software. According to the Real-Time PCR Data Markup Language (RDML) data standard (http://www.rdml.org), Cq is defined as the cycle at which the curvature of the amplification curve is maximal (fractional PCR cycles). Cq is taken in the exponential phase where the qPCR curve is linear. It is the basic result of qPCR: lower Cq values mean higher initial copy numbers of the target gene. When PCR efficiency is 100%, a difference of 1 cycle between 2 reactions means that there is 2 times more copies of the gene in the reaction that has the lower Cq value than in the reaction that has the higher Cq value.

[0055] Table 2, below, shows the metastatic invasion results, measured by qPCR for Alu sequences in the lower CAM. A metastasis regression can be seen after the treatment with bis-TPP at the lowest dose, compared to the negative control. The data show prevention of spontaneous metastasis. For the group treated at the high dose, the statistical evaluation was performed using only 3 samples surviving in the group, which impacted the statistical analysis.

Table 2. Metastasis invasion data for CAM assay.

[0056] Figure 10 illustrates the potency of bis-TPP to prevent or minimize the likelihood of tumor metastasis. As can be seen, all concentrations of 2-butene- 1,4-bis-TPP showed a near complete inhibition of tumor metastasis in the lower CAM. Thus, compounds of the present approach may be used as anti-metastasis therapeutics, as a separate therapy or in connection with other cancer therapies.

[0057] It should be appreciated that under the present approach, a therapeutic agent having an anti-mitochondrial effect, even if a side-effect or otherwise off-target property, may be used in connection with a TPP-derivative as an anti-cancer therapeutic. For example, TPP-derivatives may be administered covalently bonded with one or more therapeutic agents. The therapeutic agent may be a known pharmaceutical, including, for example, an FDA-approved antibiotic or other drug that has anti-mitochondrial side-effects. The therapeutic agent may be a mitochondrial biogenesis inhibitor, such as doxycycline, a mitoriboscin (mitoribosome-targeted therapeutics having anti-cancer and antibiotic properties), a mitoketoscin (non-carcinogenic compounds that bind to at least one of ACAT1/2 and OXCT1/2 and inhibit mitochondrial ATP production), an antimitoscin (an antibiotic having intrinsic anti-mitochondrial properties that are chemically modified to target the antibiotics to mitochondria), as additional examples. International Patent Application PCT/US2018/022403, filed March 14, 2018, International Patent Application PCT/US2018/033466, filed May 18, 2018, and International Patent Application

PCT/US2018/039354, filed September 26, 2018, are each incorporated by reference in its entirety. [0058] With respect to a mitoriboscin as a therapeutic agent, the agent may be a mitoribocycline, a mitoribomycin, a mitoribosporin, and/or a mitoribofloxin. The following compounds (or pharmaceutically acceptable salts thereof) are examples of agents that may be used: where each R may be the same or different and is selected from the group consisting of hydrogen, carbon, nitrogen, sulfur, oxygen, flourine, chlorine, bromine, iodine, carboxyl, alkanes, cyclic alkanes, alkane-based derivatives, alkenes, cyclic alkenes, alkene-based derivatives, alkynes, alkyne-based derivative, ketones, ketone -based derivatives, aldehydes, aldehyde -based derivatives, carboxylic acids, carboxylic acid-based derivatives, ethers, ether-based derivatives, esters and ester-based derivatives, amines, amino-based derivatives, amides, amide -based derivatives, monocyclic or polycyclic arene, heteroarenes, arene -based derivatives, heteroarene- based derivatives, phenols, phenol-based derivatives, benzoic acid, benzoic acid-based derivatives, and one or more mitochondrial targeting signals. For clarification, mitochondrial targeting signals are defined as any chemical or peptide entity that increases the efficiency of targeting the attached molecule to the mitochondria. Such modification would be expected to increase the potency and effectiveness of a mitoriboscin. Thus, R may be any mitochondrial targeting signal (peptide or chemical), including cationic compounds, such as tri-phenyl-phosphonium (TPP), a guanidinium- based moiety and/or choline esters, among others.

[0059] The therapeutic agent may comprise one or more of either or both an oxidative metabolism inhibitor and a glycolytic metabolism inhibitor. For example, an oxidative metabolism inhibitor may be a members of the tetracycline family and the erythromycin family. Members of the tetracycline family include tetracycline, doxycycline, tigecycline, minocycline, chlortetracycline, oxytetracycline, demeclocycline, lymecycline, meclocycline, methacycline, rolitetracycline, chlortetracycline, omadacycline, and sarecycline. Members of the erythromycin family include erythromycin, azithromycin, and clarithromycin. Inhibitors of glycolytic metabolism may be selected from inhibitors of glycolysis, inhibitors of OXPHOS, and inhibitors of autophagy. Inhibitors of glycolysis include 2-deoxy-glucose, ascorbic acid, and stiripentol. Inhibitors of OXPHOS include atoravaquone, irinotecan, sorafenib, niclosamide, and berberine chloride. Inhibitors of autophagy include chloroquine.

[0060] It should be appreciated that one or more TPP-derivatives may be the active ingredient in a pharmaceutical composition. For example, the TPP-derivative may be 2 -butene - 1,4-bis-TPP; derivatives of 2-butene- 1,4-bis-TPP; 2-chlorobenzyl-TPP (or 4-chlorobenzyl-TPP); derivatives of 2-chlorobenzyl-TPP (or 4-chlorobenzyl-TPP); 3-methylbenzyl-TPP; derivatives of 3-methylbenzyl-TPP; 2,4-dichlorobenzyl-TPP; derivatives of 2,4-dichlorobenzyl-TPP; 1- naphthylmethyl-TPP; derivatives of 1 -naphthylmethyl-TPP. Those of ordinary skill in the art will appreciate that a “derivative” of a TPP-derivative is a compound formed from the identified TPP- derivative, and may include structural analogs. The composition and/or the TPP-derivative compound may be in the form of a tablet, pill, powder, liquid, suspension, emulsion, granule, capsule, suppository, injection preparation, solution, suspension, and/or a topical cream. The pharmaceutical composition may include a therapeutic agent having an anti-mitochondrial effect. The therapeutic agent may be covalently bonded to the TPP-derivative.

[0061] In addition to bulk cancer cells and CSCs, it should be appreciated that TPP- derivative compounds may be used to target a hyper-proliferative cell sub-population that the inventors refer to as energetic cancer stem cells (e-CSCs). e-CSCs show progressive increases in sternness markers (ALDH activity and mammosphere-forming activity), highly elevated mitochondrial mass, and increased glycolytic and mitochondrial activity.

[0062] It should also be appreciated that TPP-derivative compounds may also have antibiotic and/or anti-senescence properties, among other valuable uses. For example, a TPP- derivative may be used for reducing or eliminating an age-associated illness such as atherosclerosis, cardiovascular disease, cancer, arthritis, cataracts, osteoporosis, type 2 diabetes, obesity, metabolic syndrome, hypertension, Alzheimer's disease, chronic inflammation, neuro- degeneration, muscle-wasting (sarcopenia), loss of skin elasticity, greying of the hair, male-pattern baldness, age spots, skin imperfections, and keratosis. TPP-derivatives may also be used for preventing senescence-associated secretory phenotype. In such embodiments, the TPP-derivative may be administered with a therapeutic agent having an anti-mitochondrial effect. The embodiment may take the form of a pharmaceutical composition having at least one TPP- derivative and at least on therapeutic agent having an anti-mitochondrial effect. The therapeutic agent may be covalently bonded to the TPP-derivative(s). In such embodiments, the TPP- derivative may be administered with a therapeutic agent having an anti-mitochondrial effect. The embodiment may take the form of a pharmaceutical composition having at least one TPP- derivative and at least on therapeutic agent having an anti-mitochondrial effect. The therapeutic agent may be covalently bonded to the TPP-derivative(s).

[0063] Depending on the therapeutic agent, methods and compositions as described herein may also have radiosensitizing activity, photosensitizing activity, and/or may sensitive cancer cells to one or more of chemotherapeutic agents, natural substances, and/or caloric restriction. Embodiments may also be useful for treating bacterial infection, pathogenic yeast infection, and aging. For example, the chemically modified therapeutic agent may also have enhanced anti-viral activity, enhanced anti-bacterial activity, and/or enhanced anti-microbial activity. Thus, embodiments of the present approach may also be used for targeting virus replication, preventing or reducing the growth of pathogenic bacteria, yeast, and parasites, overcoming drug resistance in bacteria (e.g., methicillin-resistant Staph. Aureus, or MSRA).

[0064] TPP-derivatives may also be used for reducing the effects of aging in an organism; treating at least one of atherosclerosis, cardiovascular disease, cancer, arthritis, cataracts, osteoporosis, type 2 diabetes, obesity, metabolic syndrome, hypertension, and Alzheimer's disease; increasing lifespan; promoting tissue repair and regeneration; and reducing aging-associated inflammation.

[0065] The group of TPP-derivative compounds described herein each inhibited CSC propagation, targeting bulk cancer cells and CSCs with minimal off-target side-effects in normal cells. By their nature, these compounds exhibit a selectivity towards mitochondria because of the presence of a lipophilic cation. The mitochondrial uptake of these compounds may be further increased through the substitution of one or more fatty acid targeting signals. A non-exhaustive list of fatty acid targeting signals includes palmitic acid, stearic acid, myristic acid, and oleic acid, a short-chain (i.e., a fatty acid with an aliphatic tail having 5 of less carbons) fatty acid, and a medium-chain (i.e., a fatty acid with an aliphatic tail having 6 to 12 carbons) fatty acid. As shown below, it should be appreciated by the person having an ordinary level of skill in the art that each TPP-derivative described herein presents more than one substitution location for a targeting signals (and other functional groups).

[0066] TPP-derivatives may be conjugated with a fatty acid targeting signal to improve the uptake and, as a result, the potency, of the TPP-derivative with respect to targeting and eradicating CSCs, and in particular, preventing or reducing the likelihood of metastasis. Compounds of the present approach have the general formula X — P ⁺ — (Ph) ₃ — Y, wherein:

X represents an organic moiety, and may be selected from carboxyls, alkanes, cyclic alkanes, alkane-based derivatives, alkenes, cyclic alkenes, alkene -based derivatives, alkynes, alkyne -based derivative, ketones, ketone-based derivatives, aldehydes, aldehyde- based derivatives, carboxylic acids, carboxylic acid-based derivatives, ethers, ether-based derivatives, esters and ester-based derivatives, amines, amino-based derivatives, amides, amide-based derivatives, monocyclic or polycyclic arene, heteroarenes, arene-based derivatives, heteroarene- based derivatives, phenols, phenol-based derivatives, benzoic acid, benzoic acid-based derivatives, and TPP-derivatives, any of which may be substituted or unsubstituted;

P ⁺ is quaternary phosphorus cation;

Ph is substituted or unsubstituted phenyl; and Y is a fatty acid targeting signal, having from 1 to 50 carbons, and more preferably from 5 to 25 carbons, and more preferably, from 10 to 20 carbons, and may be straight or branched, saturated or unsaturated, but preferably is straight and saturated.

[0067] It should be appreciated that compounds of the present approach may be salts. A targeting signal is a moiety that increases the efficiency of targeting the attached molecule to the mitochondria. Such modifications increase the potency and effectiveness of a TPP-derivative compound. In the general formula above, for example, Y may be a short-chain (i.e., a fatty acid with an aliphatic tail having 5 of less carbons) fatty acid, and a medium-chain (i.e., a fatty acid with an aliphatic tail having 6 to 12 carbons) fatty acid. In preferred embodiments, Y has a straight chain, saturated aliphatic tail, having from 10 to 20 carbons, and in some embodiments, from 11 to 16 carbons, and in some embodiments, from 12 to 15 carbons. For example, Y may be one of palmitic acid, stearic acid, myristic acid, and oleic acid. As mentioned above, the Y may be present in a conjugate as a carboacyl or an acyl amide. It should be understood by those of ordinary still in the art that Y bonded to a phenyl group will need to satisfy valence. Demonstrative examples are provided below.

[0068] Compound (I) shows a derivative 2-butene-1,4-bis-TPP with available substitution locations R ₁ -R ₆ . In this example, X is a 2-butene phosphonium, and each R-group identifies potential substitution locations for a fatty acid targeting signal, or other functional group. For example, each R may be the same or different, and may be selected from hydrogen, carbon, nitrogen, sulfur, oxygen, fluorine, chlorine, bromine, iodine, carboxyl, alkanes, cyclic alkanes, alkane-based derivatives, alkenes, cyclic alkenes, alkene-based derivatives, alkynes, alkyne-based derivative, ketones, ketone-based derivatives, aldehydes, aldehyde -based derivatives, carboxylic acids, carboxylic acid-based derivatives, ethers, ether-based derivatives, esters and ester-based derivatives, amines, amino-based derivatives, amides, amide -based derivatives, monocyclic or polycyclic arene, heteroarenes, arene-based derivatives, heteroarene- based derivatives, phenols, phenol-based derivatives, benzoic acid, benzoic acid-based derivatives, and, as mentioned above, one or more targeting signals. In preferred embodiments, one of R ₁ -R ₆ is a fatty acid targeting signal, and the remainder are H. In some preferred embodiments, the targeting signal Y is a myristate moiety.

[0069] Compound (II) shows 2-chlorobenzyl-TPP with substitution locations R ₁ -R ₄ . In this example, X is a l-chloro-2-ethylbenzyl, R ₁ is a functional location, and R ₂ -R ₄ identify potential substitution locations for a fatty acid targeting signal, or other functional group. For example, each R may be the same or different, and may be selected from hydrogen, carbon, nitrogen, sulfur, oxygen, fluorine, chlorine, bromine, iodine, carboxyl, alkanes, cyclic alkanes, alkane-based derivatives, alkenes, cyclic alkenes, alkene-based derivatives, alkynes, alkyne-based derivative, ketones, ketone-based derivatives, aldehydes, aldehyde-based derivatives, carboxylic acids, carboxylic acid-based derivatives, ethers, ether-based derivatives, esters and ester-based derivatives, amines, amino-based derivatives, amides, amide -based derivatives, monocyclic or polycyclic arene, heteroarenes, arene-based derivatives, heteroarene- based derivatives, phenols, phenol-based derivatives, benzoic acid, benzoic acid-based derivatives, and, as mentioned above, one or more fatty acid targeting signals. In preferred embodiments, one of R ₂ -R ₄ is a fatty acid targeting signal, and the remainder of R ₁ -R ₄ are H. In some preferred embodiments, the targeting signal Y is a myristate moiety. Additionally, 4-chlorobenzyl-TPP is expected to have similar, if not more potent, activity to 2-chlorobenzyl-TPP. Thus, embodiments of Compound (II) may have the halo at the 4-position, instead of the 2-position, without departing from the present approach.

[0070] Compound (III) shows 3-methylbenzyl-TPP with substitution locations R ₁ -R ₄ . In this example, X is l-ethyl-3-methylbenzyl, R ₁ is a functional location, and R ₂ -R ₄ identify potential substitution locations for a fatty acid targeting signal, or other functional group, as described above. In preferred embodiments, one of R ₂ -R ₄ is a fatty acid targeting signal, and the remainder of R ₁ -R ₄ are H. In some preferred embodiments, the targeting signal Y is a myristate moiety. [0071] Compound (IV) shows 2,4-dichlorobenzyl-TPP with substitution locations R ₁ -R ₄ .

In this example, X is 2,4-dichlorobenzyl, R ₁ is a functional location, and R ₂ -R ₄ identify potential substitution locations for a fatty acid targeting signal, or other functional group, as described above. In preferred embodiments, one of R ₂ -R ₄ is a fatty acid targeting signal, and the remainder of R ₁ -R ₄ are H. In some preferred embodiments, the targeting signal Y is a myristate moiety.

[0072] Compound (V) shows 1-naphthylmethyl-TPP with substitution locations R ₁ -R ₅ . In this example, X is naphthylmethyl, R ₄ and R ₅ are functional location, and R ₁ -R ₃ identify potential substitution locations for a fatty acid targeting signal, or other functional group, as described above. In preferred embodiments, one of R ₁ -R ₃ is a fatty acid targeting signal, and the remainder of R ₁ -R ₅ are H. In some preferred embodiments, the targeting signal Y is a myristate moiety. [0073] As another example, compound (Xa) below illustrates an embodiment of a 2- butene-1,4-bis-TPP derivative substituted with a single myristic acid targeting signal at the 4- position on one of the phenyl rings. It should be appreciated that the substitution may be made at other positions (e.g., positions 2-6) on the phenyl ring, and also on the other phenyl rings. Compound (Xb) below illustrates an embodiment of a 2-butene- 1 ,4-bis-TPP derivative substituted with a single myristic acid targeting signal, via an amide bond, at the 4-position on one of a phenyl ring. It should be appreciated that other fatty acid moieties may be used. Further, it should be appreciated that amide bonds may be used as an alternative to carboacyl bonds as shown in the various embodiments.

[0074] As a further example, compound (XI) below shows an embodiment of a 2-butene-

1,4-bis-TPP derivative substituted with two myristic acid targeting signals, each at the 4-position of phenyl rings on opposite ends of the central butane moiety. It should be appreciated that the substitutions may be made at other positions, and that more than one targeting signal may be substituted on the same side of the central butane moiety.

[0075] It should be apparent that there are several potential substitution locations and combinations that may be used according to the present approach. Efforts are underway to identify the most promising substitution locations, with respect to mitochondrial uptake and resulting efficacy in the anti-cancer therapies described herein.

[0076] Dodecyl-TPP, shown below as compound (XII) (shown in this example as a bromine salt), is a TPP-derivative that inhibits the propagation of CSCs, without undesirable, off- target effects on normal cells. In separate work, dodecyl-TPP was shown to dose-dependently inhibit breast CSC propagation in suspension. Also, dodecyl-TPP was shown to target adherent “bulk” cancer cells, by decreasing MCF-7 cell viability. Metabolic flux analysis using the Seahorse XFe96 showed that dodecyl-TPP potently inhibits the mitochondrial oxygen consumption rate (OCR), while simultaneously shifting cell metabolism toward the glycolytic pathway. As with other TPP-derivatives described herein, dodecyl-TPP may be used in combination with additional metabolic stressors as an anti-cancer therapeutic. For example, dodecyl-TPP may be combined with a glycolysis inhibitor (e.g., Vitamin C, 2-Deoxy-Glucose, etc.) and/or an OXPHOS inhibitor (e.g., Doxycycline, Niclosamide, Berberine, etc.). These combination therapies effectively decrease CSC propagation, at concentrations of dodecyl-TPP toxic only for cancer cells, but not for normal cells, as evidenced by using normal human fibroblasts (hTERT-BJ1) as a reference point.

[0077] TPP-derivatives having an alkyl chain, including for example dodecyl-TPP- derivatives, may be made through substitution of one or more hydrogens with a functional group. In some embodiments, at least one fatty acid targeting signal is substituted. Compound (XIII) shows a TPP-derivative having an alkyl chain (CH ₂ ) _n CH where n may be 0-20, with substitution locations R ₁ -R ₃ . Each R-group identifies potential substitution locations for a fatty acid targeting signal, or other functional group. For example, each R may be the same or different, and may be selected from hydrogen, carbon, nitrogen, sulfur, oxygen, fluorine, chlorine, bromine, iodine, carboxyl, alkanes, cyclic alkanes, alkane-based derivatives, alkenes, cyclic alkenes, alkene-based derivatives, alkynes, alkyne -based derivative, ketones, ketone-based derivatives, aldehydes, aldehyde -based derivatives, carboxylic acids, carboxylic acid-based derivatives, ethers, ether- based derivatives, esters and ester-based derivatives, amines, amino-based derivatives, amides, amide-based derivatives, monocyclic or polycyclic arene, heteroarenes, arene-based derivatives, heteroarene- based derivatives, phenols, phenol-based derivatives, benzoic acid, benzoic acid- based derivatives, and, as mentioned above, one or more fatty acid targeting signals. Compound (XlVa) shows an example of a TPP-derivative having a single fatty acid targeting signal and an alkyl chain, and Compound (XlVb) illustrates a TPP-derivative with a fatty acid moiety conjugated using an amide bond. Compound (XV) shows another example, in which each phenyl ring includes a fatty acid targeting signal. In these examples, if n = 11, then the TPP-derivative would be a dodecyl-TPP derivative. Of course, the fatty acid targeting signal, substitution location(s), and n for the alkyl chain, may vary from what is explicitly shown below. [0078] As used in the preceding paragraphs, the term “derivative” refers to a moiety derived from the named species, substituted with one or more functional groups. For example, it should be understood that an alkane may be a linear chain having the formula C _n H _2n+2 , or a branched chain of the formula C _n H _2n+2 in which the carbon backbone splits off in one or more directions, and a cyclic alkane may have the formula C _n H _2n (n greater than 3), in which the carbon backbone is linked to form a loop. Of course, it should be understood by those of ordinary still in the art that a substituted species bound to a phenyl group will need to satisfy valence. An alkane- based derivative is an alkane with one or more atoms, such as a hydrogen atom, substituted with a functional group such as, for example only, another alkane, an alkene, an alkyne, phenyl, amine, an alcohol, an ether, an alkyl halide, a thiol, an aldehyde, a ketone, an ester, a carboxylic acid, an amide, nitrile, epoxide, disulfide, imine, anhydride, nitro, sulfide, and so on. These are non- exhaustive examples of functional groups that may be present in a derivative compound. It should also be appreciated that the

[0079] The following paragraphs demonstrate synthesis methods for compounds of the present approach. The synthesis methods described below are intended as non-limiting examples. It should be appreciated that numerous other synthesis procedures known in the art may be used to arrive at a compound described herein, without departing from the present approach.

[0080] Substituted TPP-derivative compounds according to the present approach, having the general formula X — P ⁺ — (Ph) _{3 —} Y, may be synthesized via the following reaction schemes.

First, a monomeric aryl unit may be formed, in which the fatty acid moiety having aryl R ₇ is joined to the benzyl ring via amide bond:

[0081] In the reaction scheme shown above, W is H or (C ₁ -C ₅ )-alkyl, Y is a halogen functional group, R ₇ is one of (C ₁ -C ₅₀ )-alkyl, (C ₂ -C ₅₀ )-alkenyl, or (C ₂ -C ₅₀ )-alkynyl, and X may be an alkali or other metallic salt such as Li, Na, Mg, Al, and optionally with a halogen such as F, Cl, Br, I or without halogen. The compound of the formula (C1), e.g., haloanilines and alkylamino substituted halobenzenes, may be converted into aryl amides having the formula C2-A, through amide synthesis. A carboxylic acid moiety may be condensed to aniline by applying standard synthetic methods known in the art, such as exemplified in Chem. Soc. Rev., 2009, 38, 606-631 for the chemical transformations producing primary and secondary amides. The preparation of the compound of the formula (C2-S) with metal salt functional group Y may be performed under inert atmosphere in a reaction with a metal, such as Li, Na, or Mg, with the halogen substituent Y, preferably Cl, Br or I, of the compound with the formula of (C2-A) as exemplified in Chem. Rev. 1954, 54, 5, 835-890.

[0082] Second, the monomeric aryl unit C2-S may be used to form quaternary phosphonium compounds as shown in the following scheme:

[0083] In the reaction shown above, R ₈ , R ₉ , and R ₁₀ may be any substituent, including R ₇ amides. Compound (C7) may be the desired organic moiety X from general formula X — P ⁺ (Ph) ₃

Y, described above. The compound of the formula (C4) may be prepared by reacting the compound of formula (C2-S) and the compound of formula (C3), using standard processes known in the art for diphenylphosphane-forming reactions. For example, the preparation may be conveniently performed in the presence of base, such as potassium hydroxide or ammonia, and as exemplified in Angewandte Chemie - International Edition; vol. 56; nb. 6, p. 1643 - 1647 (2017); Journal of Organometallic Chemistry; vol. 475; nb. 1-2; p. 99 - 112 (1994); and Recueil des Travaux Chimiques des Pays-Bas; vol. Ill; nb. 4; p. 170 - 177 (1992).

[0084] The compound of formula (C4) may be converted to the compound of formula (C6) by reaction with the compound of formula (C5), where Y is a halogen (preferably bromide or iodide). The reaction may be carried out by applying the standard conditions typical to standard organometallic reactions, such as performing the reaction under an inert atmosphere, and in the presence of metal catalyst such as Pd or Ni, as exemplified in of Journal Organometallic Chemistry; vol. 866; p. 50 - 58 (2018); Tetrahedron Letters; vol. 57; nb. 30; p. 3404 - 3406 (2016); and European Journal of Organic Chemistry; vol. 2014; nb. 30; p. 6796 - 6801 (2014).

[0085] The compound of formula (C8), such as in embodiments of substituted 2-butene-

1,4-bis-TPP and 2-chlorobenzyl-TPP, may be prepared from the compound of formula (C6) by reaction with the compound of formula (C7), the X species of the general formula, which may be, as dexamples, one of alkyl halide, alkenyl halide, alkenyl dihalide, benzyl halide, substituted benzyl halide, dihalodialkyl benzene, or cyanoalkyl halide, providing the substituent Rn. The reaction may be carried out under the standard conditions for the preparation of quaternary phosphonium compounds as exemplified in Organophosphorus Compounds (1993): Phosphonium Salts, Ylides and Phosphoranes, Volume 3 (John Wiley & Sons, Ltd).

[0086] Third, the quaternary phosphonium compound of formula (C8) may be used to synthesize embodiments having a bis-TPP structural element as shown below:

[0087] The compound of formula of (C9) may be prepared reacting the compound of formula (C6) with a compound such as 1 ,4-dibromo-2-butene, under the standard conditions known in the art for the formation of phosphonium salts, for example as described in Chemische Berichte; vol. 101; nb. 4; p. 1480 - 1484 (1968). In this example, as indicated by groups R _8b -R _10b , it should be appreciated that R ₈ -R ₁₀ shown in formula (C9) do not have to be identical on each phosphonium.

[0088] These synthesis methods are demonstrative, and it should be appreciated that the person having ordinary skill in the art may use alternative synthesis methods without departing from the present approach.

[0089] The following paragraphs describe the materials and methods used in connection with the prior discussion. It should be appreciated that those having at least an ordinary level of skill in the art will be familiar with these methods.

[0090] With respect to cell culture and reagents, the human breast adenocarcinoma cell line (MCF-7) was from the American Type Culture Collection (ATCC). hTERT-BJ1 cells were from Clontech, Inc. MCF-7 and hTERT-BJ 1 cells were grown in DMEM supplemented with 10% fetal bovine serum, GlutaMAX and 1% penicillin-streptomycin and incubated at 37C in a humidified 5% C02 incubator. The medium was changed 2-3 times/week. The TPP derivatives were from Santa Cruz Biotechnology, Inc., and included: (1) 2-butene- 1,4-bis-TPP; (2) 2- chlorobenzyl-TPP; (3) 3-methylbenzyl-TPP; (4) 2,4-dichlorobenzyl-TPP; (5) 1 -naphthylmethyl- TPP; (6) mito-TEMPO; (7) cyanomethyl-TPP; (8) p-xylylene-bis-TPP; (9) 4-cyanobenzyl-TPP. [0091] With respect to the ATP-depletion assay (with CellTiter-Glo & Hoechst 33342),

MCF-7 cells were treated with different TPP derivatives for 72 hours in a black 96- well plate then wells were washed with PBS and were stained with Hoechst 33342 dye at a final concentration of 10 mg/ml. Fluorescence was read by a plate reader at 355 nm (excitation), 460 nm (emission). After washing with PBS CellTiterGlo luminescent assay (Promega) was performed according to the manufacturer’s protocols to determine intracellular ATP levels in the Hoechst dye stained cells. Both the fluorescent and luminescent data were normalized to control levels and were shown as percentage for comparison.

[0092] For measuring the mitochondrial OCR, mitochondrial function was determined by using the XF Cell Mito Stress Test Kit (Seahorse Bioscience, MA, USA) with a Seahorse XFe96 Extracellular Flux Analyzer (Seahorse Bioscience, MA, USA). MCF-7 cells were seeded in a specialized 96-well tissue culture plate (XF96 microplate). The next day, the TPP derivatives were added and the plate was incubated for 72 hours. Before the experiment media was changed to XF base medium (including 1 mM pyruvate, 2 mM glutamine and 10 mM glucose), cells were incubated at 37°C in a C02-free atmosphere for one hour before measurement. After detection of basal OCR (an indicator for mitochondrial respiration) OCR responses were evaluated towards the application of oligomycin (1 mM), FCCP (600 nM), and the combination of antimycin (1 mM) and rotenone (1 mM). From these measurements various parameters of mitochondrial function were determined. To determine cell viability in the measured wells sulphorodamine (SRB) assay was performed. Oxygen consumption rate values were then normalized to the given SRB values. [0093] For the 3D Spheroid (mammosphere) assay, a single cell suspension of MCF-7 cells was prepared using enzymatic (lx Trypsin-EDTA, Sigma Aldrich, #T3924) and manual disaggregation (25 gauge needle) to create a single cell suspension. Cells were plated at a density of 500 cells/cm2 in mammosphere medium (DMEM-F12 media including B27/20 ng/ml and EGF/PenStrep) in non-adherent conditions, in culture dishes coated with (2- hydroxyethylmethacrylate) (poly-HEMA, Sigma, #P3932). Different TPP derivatives were previously diluted in the mammosphere media before addition of cells. Plates were maintained in a humidified incubator at 37°C at an atmospheric pressure in 5% (v/v) carbon dioxide/air. After 5 days of culture, spheres >50 pm were counted using an eyepiece graticule and mammosphere numbers were normalized to control treatments (cells treated with vehicle only).

[0094] The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. The invention includes numerous alternatives, modifications, and equivalents as will become apparent from consideration of the following detailed description.

[0095] It will be understood that although the terms “first,” “second,” “third,” “a),” “b),” and “c),” etc. may be used herein to describe various elements of the invention should not be limited by these terms. These terms are only used to distinguish one element of the invention from another. Thus, a first element discussed below could be termed an element aspect, and similarly, a third without departing from the teachings of the present invention. Thus, the terms “first,” “second,” “third,” “a),” “b),” and “c),” etc. are not intended to necessarily convey a sequence or other hierarchy to the associated elements but are used for identification purposes only. The sequence of operations (or steps) is not limited to the order presented in the claims.

[0096] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the present application and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In case of a conflict in terminology, the present specification is controlling.

[0097] Also as used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0098] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed.

[0099] As used herein, the transitional phrase “consisting essentially of’ (and grammatical variants) is to be interpreted as encompassing the recited materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention. Thus, the term “consisting essentially of’ as used herein should not be interpreted as equivalent to “comprising.” [00100] The term “about,” as used herein when referring to a measurable value, such as, for example, an amount or concentration and the like, is meant to encompass variations of ± 20%, ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified amount. A range provided herein for a measureable value may include any other range and/or individual value therein.

[00101] Having thus described certain embodiments of the present invention, it is to be understood that the invention defined by the appended claims is not to be limited by particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope thereof as hereinafter claimed.