WO2009072002A2

WO2009072002A2 - Bisphenols in cancer therapy

Info

Publication number: WO2009072002A2
Application number: PCT/IB2008/003818
Authority: WO
Inventors: Brian B. Hasinoff; Hong Liang
Original assignee: University Of Manitoba
Priority date: 2007-09-14
Filing date: 2008-09-15
Publication date: 2009-06-11
Also published as: WO2009072002A3

Abstract

The present invention generally relates to anticancer agents and methods of treating cancer. The anticancer agents described herein comprise a bisphenol core and generally act as topoisomerase II inhibitors, though their anticancer activity may be related to other mechanisms as well. The bisphenols discussed herein may also protect certain cells against detrimental effects of topoisomerase II poisons.

Description

DESCRIPTION

BISPHENOLS IN CANCER THERAPY

BACKGROUND OF THE INVENTION

This application claims benefit of priority to U.S. Provisional Application Serial No. 60/993,757, filed September 14, 2007, the entire contents of which are hereby incorporated by reference.

This invention was made with government support under grant number CA90787 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention. 1. Field of the Invention

The present invention generally relates to the field of cancer treatment. More particularly, it concerns the discovery and preparation of compounds comprising a bisphenol core that are believed to exert their anticancer effects, in part, by inhibiting topoisomerase II. In another aspect of cancer therapy, the bisphenols may mitigate toxic effects of other anticancer agents via topoisomerase II inhibition.

2. Description of Related Art

The double-helical configuration of DNA strands makes them difficult to separate, and yet they must be separated by helicase proteins if other enzymes are to transcribe the sequences that encode proteins, or if chromosomes are to be replicated. Topoisomerase II (EC 5.99.1.3) is an isomerase enzyme that acts on the topology of DNA. This enzyme alters DNA topology by catalyzing the passing of an intact DNA double helix through a transient double-stranded break made in a second helix and is critical for relieving torsional stress that occurs during replication and transcription and for daughter strand separation during mitosis (Fortune and Osheroff, 2000; Li and Liu, 2001; Wang, 2002). In other words, topoisomerase Ilα cuts both DNA strands and passes an unbroken double strand through it then reanneals the cut strand — such activity is referred to as decatenation.

Mammalian cells contain α and β isoforms of topoisomerase II with topoisomerase Ilα being the most highly expressed in cells undergoing division (Akimitsu et ah, 2003). Several widely used anticancer agents, including doxorubicin, daunorubicin (and other anthracyclines), amsacrine, etoposide and mitoxantrone also target topoisomerase II and are thought to be cytotoxic because they are topoisomerase II poisons (Fortune and Osheroff, 2000; Li and Liu, 2001). Stabilization of the so-called "cleavable complex" by topoisomerase II poisons increases the levels of protein-concealed DNA double strand breaks in cells (Wilstermann and Osheroff, 2003). The action of DNA metabolic processes then renders these complexes into permanent double strand breaks, which are highly toxic to cells (Zhang et ah, 1990).

In contrast, the catalytic inhibitors of topoisomerase II do not increase the levels of DNA breaks in cells at pharmacologically relevant concentrations. There are several classes of structurally unrelated catalytic inhibitors including the bisdioxopiperazines (ICRF- 187 (dexrazoxane), ICRF- 193 and ICRF- 154), the anthracycline derivative aclarubicin, merbarone, the quinobenoxazines and novobiocin (Andoh and Ishida, 1998; Hasinoff et ah, 1995; Larsen et ah, 2003).

SUMMARY OF THE INVENTION

The present invention generally provides compounds and their use in anticancer therapies. These compounds are typically bisphenols. For example, methods of inhibiting cell growth, such as cancerous cell growth, by bisphenols are presented. Bisphenols of the present invention are also, in certain embodiments, antagonists of topoisomerase II poison-induced cleavable complex formation. This property may facilitate the use of certain bisphenols of the present invention as protective agents when administered with a topoisomerase II poison. Both cell growth inhibition and antagonism of topoisomerase II poison-induced cleavable complex formation generally relate to the novel discovery that bisphenols of the present invention are catalytic topoisomerase II inhibitors. Thus, compounds of the present invention may be utilized as anticancer agents and/or may be used in conjunction with anticancer agents.

Accordingly, certain embodiments of the present invention contemplate a method of inhibiting the catalytic decatenation activity of topoisomerase II, such as topoisomerase Ilα, comprising administering to a cell an effective amount of a bisphenol.

As used herein, "topoisomerase II" refers to both topoisomerase Ilα and topoisomerase Ilβ. In certain embodiments, only topoisomerase Ilα is contemplated. In certain embodiments, only topoisomerase Ilβ is contemplated. In certain embodiments, both topoisomerase Ilα and Ilβ are contemplated.

As used herein, the term "bisphenol" generally refers to a compound comprising two phenol groups joined by one-atom bridge, such as an ether or thioether bridge. One or both phenols may be further substituted with one or more substituents, such as a polar group, a hydrophobic group, a hydrogen bond donor, or a hydrogen bond acceptor. The atom of the one-atom bridge may be further substituted with one or more substituents (e.g., -CO-, -SO-, -SO₂-, -CH₂-, etc.).

In any method discussed herein regarding a cell, the cell may be in vivo or in vitro. The cell may be a cancer cell, and the type of cancer may be any type discussed herein.

In certain embodiments, a bisphenol of the present invention is not bisphenol A. For example, a compound of formula (I) or (II), as described below, may be further defined as a compound that is not bisphenol A.

In certain embodiments, a bisphenol of the present invention may be further defined as a compound of formula (I):

wherein: D, G, J and L are each independently -H, a polar group, a hydrophobic group, a hydrogen bond donor, or a hydrogen bond acceptor; E and K are each independently a polar group, a hydrogen bond donor, or a hydrogen bond acceptor; and P is a substituted or unsubstituted carbon or substituted or unsubstituted heteroatom (e.g., N, O, P or S). For example, certain embodiments of the present invention contemplate a method of inhibiting the catalytic decatenation activity of topoisomerase II, such as topoisomerase Ilα, comprising administering to a cell an effective amount of a bisphenol, wherein the bisphenol is a compound of formula (I). In any embodiment regarding the compound of formula (I), at least one of D, E and G may, for example, be -OH and at least one of J, K and L may, for example, be -OH. In particular embodiments regarding compounds of formula (I), D is a polar group, a hydrophobic group, a hydrogen bond donor, or a hydrogen bond acceptor; J is a polar group or a hydrogen bond donor, or a hydrogen bond acceptor; E and K are each independently a polar group, a hydrogen bond donor, or a hydrogen bond acceptor; P is an unsubstituted heteroatom; and G and L are each -H.

Regarding compounds of formula (I), the polar group of any one or more of D, G, J, L, E, or K may be, e.g., a -OH group, in certain embodiments. The hydrogen bond donor group of any one or more of D, G, J, L, E, or K may be a -OH group, in certain embodiments. The hydrogen bond acceptor of any one or more of D, G, J, L, E, or K may be a -OH or a -CHO group, in certain embodiments. P may, in certain embodiments, be a -O- or a -S- group.

In certain embodiments, any one of D, G, L, or J is a hydrogen bond acceptor, such as a polar hydrogen bond acceptor.

In certain embodiments, a bisphenol is a compound of formula (II):

wherein D, G, J, L and P are as defined above. In certain embodiments, D, G, J, L and P are further defined as the following: D and L are each independently -H, -OH, - NO₂, or an alkyl group; G and J are each independently -H, -OH, -CHO, an alkyl group, or -CH₂NRsRe, wherein R5 and R5 are each independently an alkyl group; and P is -O-, -S-, -CO-, -SO-, -SO₂-, -CH₂-, -CR₇R₈, or -C=R₉R₁₀, wherein: R₇ and R₈ are each independently -H or an alkyl group, provided that both are not H; and R₉ and R₁₀ are each independently an alkyl group or are joined together to form a cycloalkyl group. In certain embodiments, P is -O-, -S-, or -SO-.

Regarding alkyl groups of any bisphenol discussed herein, an alkyl group may, in certain embodiments, be a lower alkyl group, as defined herein. For example, the alkyl group of any one or more of D, L, G, J, R5, R5, R₇, R₈, R9, or R₁₀ of the compound of formula (II) may each independently be a lower alkyl group.

In any embodiment regarding a bisphenol, such as a compound of formula (I), the bisphenol may be further defined as any one or more of the following:

SCHO l OCHO2 O3OH

In certain embodiments, a compound of formula (I) may be further defined as

0CH02

In certain embodiments, a bisphenol of the present invention, such as a compound of formula (I), is further defined as any one or more of the following:

SCHOl OCHO2 O3OH

SCHO2 OCHOl S4OH

TDP DHDPSl

EIBP

DHDPS2

S3OH

bisphenol A bispAC3

4 ,4 -'d lhydroxybenzophenone

In certain embodiments, a bisphenol of the present invention, such as a compound of formula (I) and/or (II), is further defined as not any one or more of the following compounds:

DHDPSl BHPM

bisphenol A

EIBP 4,4-'dihydroxybenzophenone

In certain embodiments, a bisphenol of the present invention is further comprised in a pharmaceutically acceptable composition. For example, a compound of formula (I), (II), or (III), as described herein, may be comprised in a pharmaceutically acceptable composition. In certain embodiments, bisphenol A is specifically excluded as a compound that may be comprised in a pharmaceutically acceptable composition.

Other aspects of the present invention contemplated a method of treating a patient with cancer, comprising administering to the patient a therapeutically effective amount of a bisphenol, such as a compound of formula (I), (II), or (III). In certain embodiments, bisphenol A is excluded from these methods of treatment. The cancer may be, for example, cancer of the lung, liver, skin, eye, brain, gum, tongue, hematopoietic system or blood, head, neck, breast, pancreas, prostate, kidney, bone, testicles, ovary, cervix, gastrointestinal tract, lymph system, small intestine, colon, or bladder. In particular embodiments, the cancer is brain cancer.

Certain embodiments of the present invention capitalize on the antagonistic effects of a bisphenol on a topoisomerase II poison as discovered by the present inventors. For example, the present invention contemplates methods of treating malignant conditions which are sensitive to topoisomerase II poisons wherein normal tissue is substantially protected from the poison by a bisphenol such that the malignant conditions can be treated with higher dosages of the topoisomerase II poison. Accordingly, regarding certain methods of the present invention, such as a method of treating a patient with cancer (e.g., brain cancer) comprising administering to the patient a therapeutically effective amount of a bisphenol, such methods may further comprise administering a therapeutically effective amount of a topoisomerase II poison to the patient, wherein the therapeutically effective amount of the topoisomerase II poison is a cancer cell-killing amount (e.g., a brain cancer cell- killing amount) and the therapeutically effective amount of the bisphenol is a topoisomerase II poison-protective amount. A bisphenol utilized in these methods may be, in certain embodiments, S4OH, O3OH, DHDP, and/or SCHl, as these compounds are described herein.

A protective effect (e.g., a topoisomerase II poison-protective amount) of a bisphenol may be measured as described in U.S. Patent No. 6,265,385, incorporated herein by reference in its entirety, wherein bisdioxopiperazines were studied in this regard. For example, a protective effect may be shown by the increased lifespan of a mammal suffering from cancer, such as brain cancer, that has been treated with a topoisomerase II poison, such as etoposide, and a bisphenol as opposed to the lifespan of a mammal suffering from brain cancer that has been treated with the poison alone. A protective effect may also be measured by evaluating the dose of topoisomerase II poison that is administered to a patient, wherein a protective effect is shown when a maximally tolerated dosage of the poison is increased when a bisphenol is administered with the poison in a combination therapy method, as described herein, as compared to the maximally tolerated dosage of the poison in the absence of such administration of the bisphenol.

Also contemplated by the present invention are methods of reducing or preventing topoisomerase II poison-related systemic toxicity comprising administering to a subject an effective amount of a bisphenol. The bisphenol may be any bisphenol described herein. In certain embodiments, the bisphenol is further defined as S4OH, O3OH, DHDP, or SCHl, as these compounds are described herein. The subject may be a human, for example.

In embodiments wherein a bisphenol is used in topoisomerase II poison- protective amounts along with a topoisomerase II poison in cancer-cell killing amounts to treat brain cancer, the bisphenol will not cross the blood-brain barrier (BBB) while the poison will. Certain bisphenols of the present invention may cross the BBB, while others may not. Methods of measuring whether a compound crosses the BBB may be evaluated by those skilled in the art. See, e.g., Di et al., 2003; Yasmin, et al., 1988 and Koller et al., 1984, each of which is incorporated herein by reference.

The topoisomerase II poison and the bisphenol may be administered simultaneously or sequentially. By "simultaneous" administration, it is meant that topoisomerase II poison and a bisphenol are administered to a subject in a single dose by the same route of administration, or administed to a subject in two separate doses by different routes of administration but at the same time. By "sequential" it is meant that the two components are administered at different points in time, provided that the activity of the first administered agent is present and ongoing in the subject at the time the second agent is administered. For example, the bisphenol may be administered first, such that its protective effect in non-cancerous tissue outside the cancerous area {e.g. , outside the brain) is established prior to the administration of the topoisomerase II poison that targets the cancer. In certain embodiments, the poison may be administered locally while the bisphenol is administered systemically. In certain embodiments, the amount of the poison that is administered with a bisphenol is higher than what would be a pharmaceutically acceptable amount in the absence of administering a bisphenol. The amount of poison administered will typically be limited by the side effects associated with the poison. Side effects associated with topoisomerase II poisons include, for example, weight loss, leukopenia, nausea and anemia. In certain cancer treatment methods of the present invention, radiation is administered. For example, a topoisomerase II poison, a bisphenol and radiation may be administered to a subject. The order of administration may be simultaneous or sequential. For example, both the poison and the bisphenol may be administered together as radiation is commenced. In another example, the bisphenol is given first, then the poison, and then radiation. In typical embodiments, radiation is administered in the final step. Timeframes separating the administration of sequentially administered components are described in further detail below.

In certain cancer treatment methods of the present invention, the cancer is not breast cancer or leukemia. In certain embodiments, the cancer is breast cancer. In certain embodiments, the cancer is breast cancer and the bisphenol administered to treat the breast cancer is further defined as not any one or more of the following:

DHDPSl BHPM

bisphenol A

EIBP 4,4- 'dihydroxyb enzophenone

In certain embodiments, the cancer is leukemia. In certain embodiments wherein the cancer is leukemia, the bisphenol administered may, for example, be further defined as not bisphenol A.

In certain embodiments, a bisphenol of the present invention is not an endocrine disrupter. An endocrine disrupter is an agent that disrupts normal regulation of the endocrine system. Non-limiting categories of typical endocrine disrupters include estrogenic, anti-estrogenic, androgenic, anti-androgenic, thyroid hormonal and anti-thyroid hormonal agents. Non-limiting examples of endocrine disrupters include dioxin, polychlorinated biphenyls (PCBs), polybrominated biphenyls (PBBs), methoxychlor and bisphenol A. Methods of determining whether a compound is an endocrine disrupter are well-known to those of skill in the art. See, e.g., Kitamura et al., 2005 and Stroheker et al., 2004, each of which are incorporated herein by reference in its entirety. In certain embodiments, the bisphenol that is not an endocrine disrupter is further defined as a bisphenol that is not an estrogenic compound. In certain embodiments, the bisphenol is an endocrine disrupter. In embodiments where a bisphenol is an endocrine disrupter, the bisphenol may be further defined as not any one or more of the following:

In certain embodiments, a bisphenol does not induce apoptosis. In certain embodiments, the bisphenol induces apoptosis. In certain embodiments wherein a bisphenol induces apoptosis, the bisphenol may be further defined as not bisphenol A. Methods of determining whether a compound induces apoptosis are well known to those of skill in the art. See, e.g., Diel et ah, 2002 and Hansch et al, 2002, each of which is incorporated by reference in its entirety. In certain embodiments, a bisphenol of the present invention does not enhance cell proliferation and is not an apoptosis inhibitor. Methods of determining whether a compound enhances cell proliferation or is an apoptosis inhibitor are well-known to those of skill in the art. See, e.g., Diel et ah, 2002, which is incorporated herein by reference in its entirety.

In certain embodiments regarding bisphenols of formula (I), E and K of the bisphenol are both not -OH; G and J of the bisphenol are both not a 2-propenyl group; and D and L are both not hydrogen.

Other aspects of the present invention contemplate a method of antagonizing topoisomerase II poison-induced cleavable complex formation comprising administering to a cell an effective amount of a bisphenol. The bisphenol may be any bisphenol discussed herein, such as a compound of formula (I), (II), or (III), as described herein. The cell may be in vitro or in vivo. The topoisomerase II poison may be, for example, etoposide. "Antagonizing topoisomerase Ilα poison-induced cleavable complex formation" refers to the ability of a bisphenol to reduce or prevent a topoisomerase II poison from forming stabilized cleavable complexes between topoisomerase II and DNA. In certain embodiments, this phrase refers to the ability of a bisphenol to decrease the amount of linear DNA produced by a topoisomerase Ilα poison. Methods of determining whether a compound antagonizes topoisomerase II poison-induced cleavage complex formation are described in the art as well as in the Examples below. In certain embodiments, a bisphenol that antagonizes topoisomerase II poison-induced cleavable complex formation is further defined as S4OH, O3OH, DHDP, and/or SCHl, as those compounds are described herein.

Also contemplated by the present invention are therapeutic kits comprising, in suitable container means, a pharmaceutically acceptable composition comprising a bisphenol, such as a compound of formula (I), (II), or (III). In certain embodiments, bisphenol A is specifically excluded from being comprised in such kits. Such kits may be also comprise a topoisomerase II poison.

In certain embodiments, a bisphenol is a compound of formula (III):

wherein: R₁₁ is -H or -OH; and R₁₂ is -CHO or -OH; provided that when R₁₁ is -OH, Y is -SO-, and when R₁₂ is -CHO, then Y is -O-. Compounds of formula (III), or any other bisphenol described herein, may be further comprised in a pharmaceutically acceptable composition. Methods of treating a patient with cancer, comprising administering to the patient a therapeutically effective amount of a compound of formula (III), are also specifically contemplated. In this or any other method herein, the method may further comprise administering a therapeutically effective amount of a topoisomerase II poison to the patient, wherein the therapeutically effective amount of the topoisomerase II poison is a cancer cell-killing amount and the therapeutically effective amount of the bisphenol is a topoisomerase II poison-protective amount.

Other aspects of the present invention contemplate a method of inhibiting the growth of a cell, comprising administering to the cell an effective amount of a bisphenol. In certain embodiments, the bisphenol is further defined as a compound of formula (I):

wherein: D, G, J and L are each independently -H, a polar group, a hydrophobic group, a hydrogen bond donor, or a hydrogen bond acceptor; E and K are each independently a polar group, a hydrogen bond donor, or a hydrogen bond acceptor; and P is a substituted or unsubstituted carbon or a substituted or unsubstituted heteroatom; provided that at least one of D, E and G is -OH and at least one of J, K and L is -OH. In certain embodiments, the cell is a cancer cell. Non-limiting examples of cancer cells include a lung cancer cell, liver cancer cell, skin cancer cell, eye cancer cell, brain cancer cell, gum cancer cell, tongue cancer cell, hematopoietic system or blood cancer cell, head cancer cell, neck cancer cell, breast cancer cell, pancreas cancer cell, prostate cancer cell, kidney cancer cell, bone cancer cell, testicles cancer cell, ovary cancer cell, cervix cancer cell, gastrointestinal tract cancer cell, lymph system cancer cell, small intestine cancer cell, colon cancer cell, or bladder cancer cell. In particular embodiments, one or more of these types of cells are excluded from such methods. For example, the cancer cell may be further defined as neither a breast cancer cell nor a leukemia cancer cell. As discussed herein, the cell may be in vitro. In certain embodiments, the cell is in vivo.

Pharmaceutical compositions of the present invention comprise an effective amount of one or more candidate substances {e.g., a bisphenol) or additional agents dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of a pharmaceutical composition that contains at least one candidate substance or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical

Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference.

A bisphenol may be administered or delivered to a target cell, or may be administered to a subject. A bisphenol may be administered in an amount effective to treat a subject, such as a subject suffering from cancer, to produce a therapeutic benefit.

The terms "contacted" and "exposed," when applied to a cell, are used herein to describe the process by which a compound of the present invention is administered or delivered to a target cell or are placed in direct juxtaposition with the target cell. The terms "administered" and "delivered" are used interchangeably with "contacted" and "exposed."

The terms "antagonizing," "inhibiting," or "reducing" or any variation of these terms, as used herein, includes any measurable decrease or complete inhibition to achieve a desired result. For example, there may be a decrease of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more, or any range derivable therein, reduction of tumor size following administration of a bisphenol of the present invention to a cancer patient.

As used herein, the term "effective" {e.g., "an effective amount") means adequate to accomplish a desired, expected, or intended result. For example, an "effective amount" may be an amount of a compound sufficient to produce a therapeutic benefit {e.g., effective to reproducibly inhibit decrease, reduce, inhibit or otherwise abrogate the growth of a cancer cell). "Treatment" and "treating" as used herein refer to administration or application of a therapeutic agent to a subject or performance of a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit of a disease or health-related condition. For example, a subject (e.g., a mammal, such as a human) having cancer may be subjected to a treatment comprising administration of a compound of the present invention.

The term "therapeutic benefit" or "therapeutically effective" as used throughout this application refers to anything that promotes or enhances the well- being of the subject with respect to the medical treatment of a condition. This includes, but is not limited to, a reduction in the onset, frequency, or severity of the signs or symptoms of a disease. For example, a therapeutically effective amount of a compound of the present invention (e.g. , a topoisomerase II poison or a bisphenol) may be administered to a subject having a cancerous tumor, such that the tumor shrinks. Or, a therapeutically effective amount of a bisphenol of the present invention may be administered to a subject such that systemic toxicity from a topoisomerase II poison is reduced or prevented.

As used herein, the term "patient" or "subject" refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dogs, cat, mouse, rat, guinea pig, or transgenic species thereof. In certain embodiments, the patient or subject is a primate. Non-limiting examples of human subjects or patients are adults, juveniles, infants and fetuses.

As discussed herein, compounds of the present invention may, in certain embodiments, be anticancer agents. An "anticancer" agent is capable of negatively affecting cancer in a subject, for example, by killing one or more cancer cells, inducing apoptosis in one or more cancer cells, reducing the growth rate of one or more cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or one or more cancer cells, promoting an immune response against one or more cancer cells or a tumor, preventing or inhibiting the progression of a cancer, or increasing the lifespan of a subject with a cancer. Various anticancer agents are well-known in the art and include, for example, chemotherapy agents (chemotherapy), such as DNA intercalators, radiotherapy agents (radiotherapy), a surgical procedure, immune therapy agents (immunotherapy), genetic therapy agents (gene therapy), reoviral therapy, hormonal therapy, other biological agents (biotherapy), and/or alternative therapies.

A cell-killing amount of a compound of the present invention, such as a cancer cell-killing amount, is an amount of compound that results in the killing of at least one cell, To kill a cell in accordance with the present invention, one would generally contact the cell with a compound of the present invention in amount effective to kill the cell. The term "in an amount effective to kill the cell" means that the amount of the compound of the present invention is sufficient so that, when administered to a cell, cell death is induced. A number of in vitro parameters may be used to determine the effect produced by the compositions and methods of the present invention. These parameters include, for example, the observation of net cell numbers before and after exposure to the compositions described herein.

A "topoisomerase II poison," as used herein, refers to an agent that stabilizes a topoisomerase II enzyme-DNA cleavable complex and shifts the equilibrium of the catalytic cycle towards cleavage, thereby increasing the concentration of the transient protein-associated breaks in the genome. Methods of determining whether an agent is a poison are known in the art, and certain methods are described herein. The topoisomerase may be topoisomerase Ilα, topoisomerase Ilβ, or both. Non-limiting examples of such poisons include etoposide, teniposide, m-AMSA (4'-(9- acridinylamino)methanesulfone-m-anisidide), daunorubicine and mitoxantrone, and salts thereof.

Moreover, it is specifically contemplated that definitions or descriptions of compounds may be further defined to exclude any compound or class of compounds discussed herein. Indeed, any specific compound or genus may be excluded from any embodiment herein. For example, any one or more of the following compounds may be excluded from embodiments discussed herein, such as methods of treating cancer or methods of inhibiting the growth of a cell, such as a cancer cell:

SCHOl OCHO2 O3OH

SCHO2 OCHOl

S4OH

TDP DHDPSl

EIBP

DHDPS2

S3OH

bisphenol A bispAC3

4,4-'dihydroxybenzophenone

It is also specifically contemplated that any limitation discussed with respect to one embodiment of the invention may apply to any other embodiment of the invention. Furthermore, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention.

The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternative are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or."

Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the device and/or method being employed to determine the value. As used herein the specification, "a" or "an" may mean one or more, unless clearly indicated otherwise. As used herein in the claim(s), when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more than one. As used herein "another" may mean at least a second or more.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. The patent or application file contains at least one drawing executed in color.

Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIG. 1. Structures of certain bisphenols tested for inhibition of topoisomerase Ilα and inhibition of cell growth. FIG. 2. The bisphenols S4OH and DHDP inhibit K562 cell growth and the catalytic decatenation activity of topoisomerase Ilα. FIG. 2A: Inhibition of growth of K562 cells by S4OH (o) and DHDP(«). Cells were treated with the bisphenol indicated for 72 h prior to assessment of growth inhibition by an MTS assay. The curved solid lines are non-linear least squares fits to a 4-parameter logistic equation and yield /C₅₀ values of 64 ± 24 and 9.7 ± 5.5 μM, respectively for S4OH and DHDP. FIG. 2B: Inhibition of the catalytic decatenation activity of topoisomerase Ilα by S4OH (o) and DHDP (•) The curved solid lines are non-linear least squares fits to a 4-parameter logistic equation and yield ICso values of 0.69 ± 0.07 and 31 ± 12 μM, respectively for S4OH and DHDP.

FIG. 3. Correlations of growth inhibition of CHO and K562 cells with inhibition of topoisomerase Ilα by the bisphenols. FIG. 3A: Correlation of the IC^ for growth inhibition of CHO cells with the IC^ for the inhibition of topoisomerase Ilα. FIG. 3B: Correlation of the /C50 for growth inhibition of K562 cells with the /C50 for the inhibition of topoisomerase Ilα. The data is plotted on both axes with logarithmic scales. The straight lines are linear least squares calculated.

FIG. 4. Effect of bisphenols on the topoisomerase Ilα-mediated relaxation and cleavage of supercoiled pBR322 DNA and their inhibition of etoposide -induced DNA cleavage. FIG. 4A: This fluorescent image of the ethidium bromide-stained gel shows that none of the bisphenols produced linear DNA above control levels (lane 5). As shown in lane 7 etoposide treatment produced linear DNA (LIN). In this gel the relaxed DNA (RLX) ran only slightly ahead of the supercoiled DNA. None of the bisphenols tested produced detectable amounts of linear DNA, indicating that they were not topoisomerase Ilα poisons. Topoisomerase Ilα was present in the reaction mixture in all lanes but lane 6. A small amount of nicked circular (NC) is normally present in the pBR322 DNA. The bisphenols and etoposide were present at 250 μM. Topo Ilα is topoisomerase Ilα. FIG. 4B: This fluorescent image of the ethidium bromide-stained gel shows that based on the decrease in the integrated intensity of the linear pBR322 DNA band, relative to that of the etoposide-alone treatment (lane 6), all of the bisphenols partially antagonized etoposide-induced formation of linear DNA. This gel also shows that all of the bisphenols inhibited the relaxation of supercoiled DNA. Where indicated the bisphenols and etoposide were present at 100 μM. FIG. 4C: Bar graph of the integrated band intensities for the linear DNA band for the compounds tested in B above. Etoposide produced a 5.2-fold increase in linear DNA (lane 6) compared to the no-drug control (lane 5). All of the bisphenols decreased the amount of linear DNA produced by etoposide by amounts ranging from 29 to 84 %, indicating that they antagonized etoposide-induced linear DNA formation.

FIG. 5. Effect of preincubation of K562 cells with bisphenol topoisomerase Ilα catalytic inhibitors. FIG. 5A: K562 cells were either untreated (o) or pretreated (•) with 5 μM O3OH for 30 min prior to treatment with doxorubicin for 72 h prior to assessment of growth inhibition by an MTS assay. The curved solid lines are nonlinear least squares fits to a 4-parameter logistic equation and yield /C50 values of 0.065 ± 0.022 and 0.142 ± 0.008 μM, respectively, for no pretreatment and pretreatment with 5 μM O3OH. FIG. 5B: K562 cells were either untreated (o) or pretreated (•) with 5 μM S4OH for 30 min prior to treatment with doxorubicin for 72 h prior to assessment of growth inhibition by an MTS assay. The curved solid lines are non-linear least squares fits to a 4-parameter logistic equation and yield an /C50 of 0.0017 ± 0.006 μM for pretreatment with 5 μM O3OH. The concentration of 5 μM of O3OH or S4OH are about 6-fold higher than that required to inhibit the catalytic activity of topoisomerase Ilα. Neither O3OH nor S4OH antagonized the growth inhibitory effects of doxorubicin. FIG. 5C: K562 cells were either untreated (o) or pretreated (•) with 300 μM DHDP for 30 min prior to a 1 h treatment with etoposide after which both drugs were washed off. After 72 h growth inhibition was assessed by an MTS assay. The curved solid lines are non-linear least squares fits to a logistic equation and yield IC50 values of 17 ± 11 and 97 ± 22 μM, respectively, for no pretreatment and pretreatment with 300 μM DHDP. FIG. 5D: K562 cells were either untreated (o) or pretreated (•) with 300 μM S4OH for 30 min prior to a 1 h treatment with etoposide. After 72 h growth inhibition was assessed by an MTS assay. The curved solid lines are non-linear least squares fits to a 4-parameter logistic equation and yield an /C₅₀ of 12 ± 10 μM for pretreatment with 300 μM S4OH. The concentration of 300 μM of DHDP or S4OH were much higher than that required to inhibit the catalytic activity of topoisomerase Ilα.

FIG. 6. FIG. 6A: Structure of OCHO2 that was used as a template molecule in the 3D-QSAR modeling for the inhibition of topoisomerase Ilα. The atoms connecting the bonds in bold were used for the molecular alignments in the CoMFA and CoMSIA analyses. FIG. 6B: Structures of 23 bisphenol energy-minimized structures aligned to the template molecule OCHO2 used in the 3D-QSAR modeling. FIG. 6C: Electrostatic, FIG. 6D: H-bond acceptor, FIG. 6E: H-bond donor and FIG. 6F: hydrophobic stddev*coeff contour maps superimposed on the structure of OCHOH2 obtained from the CoMSIA modeling for the topoisomerase Ilα inhibitory activity of 18 bisphenols. In this order, these were the four most important field components that resulted from the CoMSIA modeling. The green grids outline the regions in space for each field that were favored for topoisomerase Ilα inhibition, while the red areas show the regions that were disfavored.

FIG. 7 A and 7B: Correlation of the predicted and experimentally determined values ofpICso for inhibition of the decatenation activity of topoisomerase Ilα by the bisphenol analogs used in the building of the CoMFA and CoMSIA models, respectively. FIG. 7C and 7D: Correlation of the predicted and experimentally determined values of pIC ₅₀ for inhibition of the K562 cell growth by the bisphenol analogs used in the building of the CoMFA and CoMSIA models, respectively. The straight lines were linear least squares calculated.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present inventors have discovered a novel class of topoisomerase II inhibitors. These compounds are bisphenols or derivatives thereof. Due at least in part to their cell growth inhibition activity, compounds of the present invention are, in certain embodiments, candidate anticancer agents. Additionally, certain bisphenols of the present invention may provide protection against systemic toxicity induced by the administration of a topoisomerase II poison. Certain bisphenols of the present invention are novel as well.

A. Bisphenol Anticancer Activity

The present inventors have shown that bisphenols potently inhibit the growth of CHO and K562 cells in the low micromolar range. The positive correlation of cell growth inhibition of CHO and K562 cells with topoisomerase Ilα inhibition (p < 0.001 and 0.02, respectively, and an r² of 0.56 and 0.24, respectively) suggests that the catalytic inhibition of topoisomerase II contributes to the growth inhibitory activity, even though other mechanisms may be in play. This correlation can be compared to a previous QSAR study in which a series of 12 bisdioxopiperazines, that included dexrazoxane (ICRF-187) and ICRF-193, was examined, showing that the logarithm of the CHO /C50 was correlated with the catalytic inhibition of topoisomerase II with an r² = 0.74 and a p of 0.0003 (Hasinoff et al, 1995). As a group the bisdioxopiperazines may only target topoisomerase II, whereas the bisphenols may also have inhibited cell growth through other mechanisms involving other targets and also possibly through cytotoxic metabolites.

The fact that certain compounds of the present invention did not induce topoisomerase Ilα-mediated cleavage of DNA and were not cross resistant to the KTVP.5 cell line with a reduced level of topoisomerase Ilα (Fattman et al., 1996; Ritke et al, 1994) indicates that these compounds do not act as topoisomerase II poisons, and thus are pure catalytic inhibitors of topoisomerase Ilα. Topoisomerase II catalytic inhibitors such as the bisdioxopiperazines dexrazoxane and ICRF- 193 (Hasinoff et al., 1996; Sehested et al., 1993) and a newly identified "purine class" (NSC35866) of compounds (Jensen et al., 2005) can antagonize the growth inhibitory effects of topoisomerase II poisons. However, not all of the purines acted like NSC35866 as a follow-up study by the present inventors showed that most of the purines were not capable of antagonizing etoposide-induced cytotoxicity and DNA strand breaks in cells (Jensen et al., 2006). S4OH, O3OH, DHDP, and SCHl antagonized etoposide-induced linear DNA formation by purified topoisomerase Ilα (FIGs. 4B and 4C). However, the bisphenols O3OH and S4OH do antagonize the K562 cell growth inhibitory effects of the topoisomerase II poison doxorubicin or etoposide (FIGs. 5A, 5B and 5D) though, in contrast, DHDP did antagonize the growth inhibitory effects of etoposide (FIG. 5C). It may be that there are subtle differences in the way that the bisphenols interact with topoisomerase II. In the case of doxorubicin it may also have exerted its growth inhibitory effects by other mechanisms such as reactive oxygen species formation (Gewirtz, 1999). The lack of cross resistance (Table 1) with the bisdioxopiperazine-resistant DZR cell line indicates that in spite of some general structural similarities, that the bisphenols did not bind at the bisdioxopiperazine binding site of topoisomerase Ilα.

Table 1

Cell growth inhibitory and topoisomerase II inhibitory effects of certain bisphenols

a The relative resistance factor RR was calculated from the ratio of the /C₅₀ value for the K/VP.5 cell line divided by that for the K562 cell line or the DZR to the CHO cell line. ND is not determined.

In order to further define the structural factors that result in high cell growth inhibitory potency and topoisomerase Ilα inhibitory activity for the bisphenols, 3D- QSAR CoMFA and CoMSIA analyses were carried out in order to derive a model for the prediction of activity to aid in the synthesis of new and more active analogs. Based on a common substructure alignment (FIGs. 6A and 6B), only the CoMSIA analyses for the topoisomerase Ilα inhibitory activity gave a high quality models based on their q² values (Table 2). The largest contributors to the CoMSIA field were electrostatic, hydrogen bond acceptor, hydrogen bond donor, hydrophobic and steric contributions, in that order. Mapping of these field onto the structure of OCHO2, one of the most cytotoxic bisphenols, showed that for both the electrostatic and hydrogen bond acceptor fields bisphenol analogs with an hydroxyl or formyl groups in the meta position on the phenyl rings there was a favorable contribution to the topoisomerase Ilα inhibitory activity. Polar meta hydrogen bond acceptor substituents on the phenyl rings also favored inhibition of topoisomerase Ilα.

Table 2. Partial least-squares statistics and field contributions from CoMFA and CoMSIA models for the prediction of pICso for the inhibition of topoisomerase

Ilα

q : Leave -one -out (LOO) cross-validated correlation coefficient N: optimum number of components r²: non-cross-validated correlation coefficient

SEE: standard error of estimate

F: i^-test value

SEP: standard error of prediction B. Protective Effects of Bisphenols

As mentioned above, certain bisphenols of the present invention were found by the present inventors to antagonize etoposide-induced linear DNA formation by purified topoisomerase Ilα (FIGs. 4B and 4C). This effect may be utilized to mitigate toxic effects seen with topoisomerase II poisons such as etoposide. Topoisomerase II poisons such as etoposide are usually already used in maximally tolerated doses in the clinic; therefore, dose increments which otherwise might have overcome drug resistance are not feasible. However, use of an antagonist together with an agonist according to certain embodiments of the present invention may yield new prospects. Ideally, manipulation of the effects of topoisomerase II poisons should permit significant dose escalations in vivo. For example, a topoisomerase II poison may be administered to a subject, e.g., to a human, in an amount that kills one or more cancer cell of interest (e.g. , cancer cell in a tumor, or metastazised cancer) together with administration of a bisphenol. The non- cancerous cells (e.g., tissues) in the subject may preferentially protected against the toxic action of the topoisomerase II poison by the bisphenol, whereby increased dosages of the topoisomerase II poison are tolerated compared to the conventional administration of the topoisomerase II poison alone. A bisphenol may be used in a preventative context in this regard by being administered before administration or exposure to a topoisomerase II poison, or may be administered simultaneously or after the administration of a poison (see Combination Therapy, below).

Such protection may be especially useful when a topoisomerase II poison is used to treat a cancer of the central nervous system (e.g., the brain). The poison may cross the blood brain barrier (BBB) while a bisphenol may be chosen that does not, such that the bisphenol can protect against or reduce any systemic toxicity caused by the poison.

C. Chemical Definitions

As used herein, the term "amino" means -NH₂; the term "nitro" means -NO₂; the term "halo" designates -F, -Cl, -Br or -I; the term "mercapto" or "thiol" means -SH; the term "cyano" means -CN; the term "azido" means -N₃; the term "silyl" means -SiH₃, and the term "hydroxy" means -OH.

The term "alkyl" includes straight-chain alkyl, branched-chain alkyl, cycloalkyl (alicyclic), cyclic alkyl, heteroatom-unsubstituted alkyl, heteroatom- substituted alkyl, heteroatom-unsubstituted C_n-alkyl, and heteroatom-substituted C_n-alkyl. In certain embodiments, lower alkyls are contemplated. The term "lower alkyl" refers to alkyls of 1-6 carbon atoms (that is, 1, 2, 3, 4, 5 or 6 carbon atoms, or any range derivable therein). The term "heteroatom-unsubstituted C_n-alkyl" refers to a radical, having a linear or branched, cyclic or acyclic structure, further having no carbon-carbon double or triple bonds, further having a total of n carbon atoms, all of which are nonaromatic, 3 or more hydrogen atoms, and no heteroatoms. For example, a heteroatom-unsubstituted Ci-Cio-alkyl has 1 to 10 carbon atoms. The groups, -CH₃ (Me), -CH₂CH₃ (Et), -CH₂CH₂CH₃ (n-Pr), -CH(CH₃)₂ (iso-Pr), -CH(CH₂)₂ (cyclopropyl), -CH₂CH₂CH₂CH₃ (n-Bu), -CH(CH₃)CH₂CH₃ (sec-butyl), -CH₂CH(CHs)₂ (wo-butyl), -C(CH₃)₃ (tert-butyϊ), -CH₂C(CH₃)₃ (neo-pentyl), cyclobutyl, cyclopentyl, and cyclohexyl, are all non-limiting examples of heteroatom- unsubstituted alkyl groups. The term "heteroatom-substituted C_n-alkyl" refers to a radical, having a single saturated carbon atom as the point of attachment, no carbon- carbon double or triple bonds, further having a linear or branched, cyclic or acyclic structure, further having a total of n carbon atoms, all of which are nonaromatic, 0, 1, or more than one hydrogen atom, at least one heteroatom, wherein each heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S. For example, a heteroatom-substituted Ci-Cio-alkyl has 1 to 10 carbon atoms. The following groups are all non- limiting examples of heteroatom-substituted alkyl groups: trifluoromethyl, -CH₂F, -CH₂Cl, -CH₂Br, -CH₂OH, -CH₂OCH₃, -CH₂OCH₂CF₃, -CH₂OC(O)CH₃, -CH₂NH₂, -CH₂NHCH₃, -CH₂N(CH₃)₂, -CH₂CH₂Cl, -CH₂CH₂OH, CH₂CH₂OC(O)CH₃, -CH₂CH₂NHCO₂C(CH₃)₃ and -CH₂Si(CH₃)₃. In certain embodiments, unsubstituted alkyl groups are contemplated. The term "alkoxy" when used without the "substituted" modifier refers to the group -OR, in which R is an alkyl, as that term is defined above. Non-limiting examples of alkoxy groups include: -OCH₃, -OCH₂CH₃, -OCH₂CH₂CH₃, -OCH(CH₃)₂, -OCH(CH₂)₂, -O-cyclopentyl, and -O-cyclohexyl. The term "substituted alkoxy" refers to the group -OR, in which R is a substituted alkyl, as that term is defined above. For example, -OCH₂CF₃ is a substituted alkoxy group.

Modifications or derivatives of the compounds, agents, and active ingredients disclosed throughout this specification are contemplated as being useful with the methods and compositions of the present invention. Derivatives may be prepared and the properties of such derivatives may be assayed for their desired properties by any method known to those of skill in the art.

In certain aspects, "derivative" refers to a chemically modified compound that still retains the desired effects of the compound prior to the chemical modification. "Bisphenol derivatives," therefore, refer to a chemically modified compound that still retains the desired effects of the parent bisphenol prior to its chemical modification. Such effects may be enhanced (e.g., slightly more effective, twice as effective, etc.) or diminished (e.g., slightly less effective, 2-fold less effective, etc.) relative to he parent bisphenol, but may still be considered a bisphenol derivative. Such derivatives may have the addition, removal, or substitution of one or more chemical moieties on the parent molecule. Non-limiting examples of the types modifications that can be made to the compounds and structures disclosed herein include the addition or removal of lower unsubstituted alkyls such as methyl, ethyl, propyl, or substituted lower alkyls such as hydroxymethyl or aminomethyl groups; carboxyl groups and carbonyl groups; hydroxyls; nitro, amino, amide, and azo groups; sulfate, sulfonate, sulfono, sulfhydryl, sulfonyl, sulfoxido, phosphate, phosphono, phosphoryl groups, and halide substituents. Additional modifications can include an addition or a deletion of one or more atoms of the atomic framework, for example, substitution of an ethyl by a propyl; substitution of a phenyl by a larger or smaller aromatic group. Alternatively, in a cyclic or bicyclic structure, heteroatoms such as N, S, or O can be substituted into the structure instead of a carbon atom.

Prodrugs and solvates of the compounds of the present invention are also contemplated herein. The term "prodrug" as used herein, is understood as being a compound which, upon administration to a subject, such as a mammal, undergoes chemical conversion by metabolic or chemical processes to yield a compound any of the formulas herein, or a salt and/or solvate thereof (Bundgaard, 1991; Bundgaard, 1985). Solvates of the compounds of the present invention may be hydrates.

The term "pharmaceutically acceptable salts," as used herein, refers to salts of compounds of this invention that are substantially non-toxic to living organisms. Typical pharmaceutically acceptable salts include those salts prepared by reaction of a compound of this invention with an inorganic or organic acid, or an organic base, depending on the substituents present on the compounds of the invention.

Non-limiting examples of inorganic acids which may be used to prepare pharmaceutically acceptable salts include: hydrochloric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid and the like. Examples of organic acids which may be used to prepare pharmaceutically acceptable salts include: aliphatic mono- and dicarboxylic acids, such as oxalic acid, carbonic acid, citric acid, succinic acid, phenyl-heteroatom-substituted alkanoic acids, aliphatic and aromatic sulfuric acids and the like. Pharmaceutically acceptable salts prepared from inorganic or organic acids thus include hydrochloride, hydrobromide, nitrate, sulfate, pyrosulfate, bisulfate, sulfite, bisulfate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, hydroiodide, hydrofluoride, acetate, propionate, formate, oxalate, citrate, lactate, p-toluenesulfonate, methanesulfonate, maleate, and the like. Suitable pharmaceutically acceptable salts may also be formed by reacting the agents of the invention with an organic base such as methylamine, ethylamine, ethanolamine, lysine, ornithine and the like.

Pharmaceutically acceptable salts include the salts formed between carboxylate or sulfonate groups found on some of the compounds of this invention and inorganic cations, such as sodium, potassium, ammonium, or calcium, or such organic cations as isopropylammonium, trimethylammonium, tetramethylammonium, and imidazolium. Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, Selection and Use (P. H. Stahl & C. G. Wermuth eds., Verlag Helvetica Chimica Acta, 2002), which is incorporated herein by reference.

The term "functional group" generally refers to how persons of skill in the art classify chemically reactive groups. Examples of functional groups include hydroxyl, amine, sulfhydryl, amide, carboxyls, carbonyls, etc. As used herein, "protecting group" refers to a moiety attached to a functional group to prevent an otherwise unwanted reaction of that functional group. Protecting groups are well-known to those of skill in the art. Non-limiting exemplary protecting groups fall into categories such as hydroxy protecting groups, amino protecting groups, sulfhydryl protecting groups and carbonyl protecting groups. Such protecting groups may be found in Greene and Wuts, 1999. Bisphenols comprising various protecting groups are specifically contemplated by the present invention.

As used herein, a "hydrogen bond" refers to the primarily electrostatic bond formed by the interaction of a hydrogen atom covalently bound to an electronegative atom {e.g., oxygen, nitrogen, or fluorine) and a second electronegative atom. The bonding partners consist of a "hydrogen bond donor atom," (that is the atom to which hydrogen is covalently bound), and the "hydrogen bond acceptor atom." Electronegative atoms, as used herein, refer to atoms that are more electronegative than hydrogen (otherwise there would be no energetically favorable reason for the hydrogen attached to a donor atom to interact with the acceptor atom). One can estimate the strength of such bonds, as it is well-known that the greater the difference in electronegativity values between hydrogen and another atom, the greater the potential for that atom to behave as a hydrogen bond donor. Tables of such values are easily accessed by skilled artisans. See, e.g., Zumdahl, (1993), pp 345-347. One can also directly measure the strength of hydrogen bonds using methods well-known to those of skill in the art (e.g., x-ray and neutron diffraction). Non-limiting examples of hydrogen bond donor groups include hydroxy, -CHO, -SH, -NH₂ and -NHR groups. Hydrogen bond acceptors typically have at least one nonbonding pair of electrons. Non-limiting examples of hydrogen bond acceptor groups include hydroxy, -CHO and other carboxyl groups, -NH₂, alkoxy, alkylthio and nitro groups. In addition, a hydrogen bond donor may be a polar hydrogen bond donor. Similarly, a hydrogen bond acceptor may be a polar hydrogen bond acceptor.

"Polar groups" are well-known to those of skill in the art and typically refer to a group comprising two covalently bound atoms of differing electronegativity, such that the electrons between two atoms are shared unevenly. Methods of determining electronegativities are described above. A polar group has an overall polarity that is not cancelled by, e.g., the shape of the group. Non-limiting examples of polar groups include hydroxy, methoxy, dimethylamino, -CHO and trifluoromethyl.

"Hydrophobic groups" are well-known to those of skill in the art and are water-insoluble groups or exhibit low water solubility. In certain embodiments, "low water-solubility" refers to a solubility of a compound in water at 25 ⁰C of about or less than about 100 mg/ml, such as about or less than about 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 7.5, 5, 2.5, 1, 0.5, or 0.1 mg/ml, or lower, or any range derivable therein. Methods of measuring water solubility are well-known in the art. Hydrophobic groups are typically non-polar, but not necessarily. Non-limiting examples of hydrophobic groups include unsubstituted straight-chain, branched or cyclic alkyl groups or aromatic groups comprising only hydrogen and carbon (e.g., ethyl, t-butyl, propenyl, phenyl) as well as related unsubstituted alkoxy groups.

Compounds of the present invention may contain one or more asymmetric centers and thus can occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. In certain embodiments, a single diastereomer is present. All possible stereoisomers of the compounds of the present invention are contemplated as being within the scope of the present invention. However, in certain aspects, particular diastereomers are contemplated. The chiral centers of the compounds of the present invention can have the S- or the R- configuration, as defined by the IUPAC 1974 Recommendations. In certain aspects, certain compounds of the present invention may comprise S- or ^-configurations at particular carbon centers. Solvent choices for the methods of the present invention will be known to one of ordinary skill in the art. Solvent choices may depend, for example, on which one(s) will facilitate the solubilizing of all the reagents or, for example, which one(s) will best facilitate the desired reaction (particularly when the mechanism of the reaction is known). Solvents may include, for example, polar solvents and non-polar solvents. Solvents choices include, but are not limited to, tetrahydrofuran, dimethylformamide, dimethylsulfoxide, dioxane, methanol, ethanol, hexane, methylene chloride and acetonitrile. More than one solvent may be chosen for any particular reaction or purification procedure. Water may also be admixed into any solvent choice. Further, water, such as distilled water, may constitute the reaction medium instead of a solvent.

Persons of ordinary skill in the art will be familiar with methods of purifying compounds of the present invention. One of ordinary skill in the art will understand that compounds of the present invention can generally be purified at any step, including the purification of intermediates as well as purification of the final products. In certain embodiments, purification is performed via silica gel column chromatography or HPLC.

In view of the above definitions, other chemical terms used throughout this application can be easily understood by those of skill in the art. Terms may be used alone or in any combination thereof. D. Pharmaceutical Preparations

Certain of the methods set forth herein pertain to methods involving the administration of a pharmaceutically effective amount of a bisphenol for chemotherapeutic purposes. In certain embodiments, the bisphenols of this invention may be administered to kill tumor cells by any method that allows contact of the active ingredient with the agent's site of action in the tumor. They may be administered by any conventional method available for use in conjunction with pharmaceuticals, either as individual therapeutically active ingredients or in a combination of therapeutically active ingredients. They may be administered alone, but are generally administered with a pharmaceutically acceptable carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.

The bisphenols may be extensively purified and/or dialyzed to remove undesired small molecular weight molecules and/or lyophilized for more ready formulation into a desired vehicle, where appropriate. Such methods are well-known in the art. The active compounds will then generally be formulated for administration by any known route, such as parenteral administration. Methods of administration are discussed in greater detail below. Aqueous compositions of the present invention will typically have an effective amount of a bisphenol to kill or slow the growth of cancer cells. Further, the potential recognition of genes can be accomplished by the synthesis of bisphenols with specific structures that allow for the recognition of specific parts of DNA. Such compositions will generally be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

Moreover, it will be generally understood that any bisphenol can be provided in prodrug form, meaning that an environment to which a bisphenol is exposed alters the prodrug into an active, or more active, form. It is contemplated that the term "precursor" covers compounds that are considered "prodrugs." 1. Pharmaceutical Formulations and Routes for Administration

Pharmaceutical compositions of the present invention comprise an effective amount of one or more candidate substances (e.g., a bisphenol) or additional agents dissolved or dispersed in a pharmaceutically acceptable carrier. The phrase "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of a pharmaceutical composition that contains at least one candidate substance or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, pp 1289-1329, 1990). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

The candidate substance may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection. Compounds of the present invention may be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, locally, via inhalation (e.g., aerosol inhalation), via injection, via infusion, via continuous infusion, via localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the foregoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 1990).

In particular embodiments, the composition is administered to a subject using a drug delivery device. Any drug delivery device is contemplated for use in delivering a pharmaceutically effective amount of a bisphenol. The actual dosage amount of a composition of the present invention administered to an animal patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

The dose can be repeated as needed as determined by those of ordinary skill in the art. Thus, in some embodiments of the methods set forth herein, a single dose is contemplated. In other embodiments, two or more doses are contemplated. Where more than one dose is administered to a subject, the time interval between doses can be any time interval as determined by those of ordinary skill in the art. For example, the time interval between doses may be about 1 hour to about 2 hours, about 2 hours to about 6 hours, about 6 hours to about 10 hours, about 10 hours to about 24 hours, about 1 day to about 2 days, about 1 week to about 2 weeks, or longer, or any time interval derivable within any of these recited ranges.

In certain embodiments, it may be desirable to provide a continuous supply of a pharmaceutical composition to the patient. This could be accomplished by catheterization, followed by continuous administration of the therapeutic agent. The administration could be intra-operative or post-operative.

In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of a bisphenol. In other embodiments, the bisphenol may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. In other non- limiting examples, a dose may also comprise from about 1, 5, 10, 50, 100, 200, 350, or 500 microgram/kg/body weight, about 1, 5, 10, 50, 100, 200, 350, 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc., can be administered, based on the numbers described above.

In any case, the composition may comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens {e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof. The candidate substance may be formulated into a composition in a free base, neutral, or salt form. Pharmaceutically acceptable salts, include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine, or procaine.

In embodiments where the composition is in a liquid form, a carrier can be a solvent or dispersion medium comprising but not limited to, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes) and combinations thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin; by the maintenance of the required particle size by dispersion in carriers such as, for example liquid polyol or lipids; by the use of surfactants such as, for example hydroxypropylcellulose; or combinations thereof such methods. It may be preferable to include isotonic agents, such as, for example, sugars, sodium chloride, or combinations thereof.

In other embodiments, one may use eye drops, nasal solutions or sprays, aerosols or inhalants in the present invention. Such compositions are generally designed to be compatible with the target tissue type. In a non- limiting example, nasal solutions are usually aqueous solutions designed to be administered to the nasal passages in drops or sprays. Nasal solutions are prepared so that they are similar in many respects to nasal secretions, so that normal ciliary action is maintained. Thus, in certain embodiments the aqueous nasal solutions usually are isotonic or slightly buffered to maintain a pH of about 5.5 to about 6.5. In addition, antimicrobial preservatives, similar to those used in ophthalmic preparations, drugs, or appropriate drug stabilizers, if required, may be included in the formulation. For example, various commercial nasal preparations are known and include drugs such as antibiotics or antihistamines.

In certain embodiments the candidate substance is prepared for administration by such routes as oral ingestion. In these embodiments, the solid composition may comprise, for example, solutions, suspensions, emulsions, tablets, pills, capsules (e.g., hard or soft shelled gelatin capsules), sustained release formulations, buccal compositions, troches, elixirs, suspensions, syrups, wafers, or combinations thereof. Oral compositions may be incorporated directly with the food of the diet. In certain embodiments, carriers for oral administration comprise inert diluents, assimilable edible carriers or combinations thereof. In other aspects of the invention, the oral composition may be prepared as a syrup or elixir. A syrup or elixir, and may comprise, for example, at least one active agent, a sweetening agent, a preservative, a flavoring agent, a dye, a preservative, or combinations thereof.

In certain embodiments an oral composition may comprise one or more binders, excipients, disintegration agents, lubricants, flavoring agents, or combinations thereof. In certain embodiments, a composition may comprise one or more of the following: a binder, such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; an excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or combinations thereof; a disintegrating agent, such as, for example, corn starch, potato starch, alginic acid or combinations thereof; a lubricant, such as, for example, magnesium stearate; a sweetening agent, such as, for example, sucrose, lactose, saccharin or combinations thereof; a flavoring agent, such as, for example peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.; or combinations thereof the foregoing. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, carriers such as a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar, or both.

Additional formulations which are suitable for other modes of administration include suppositories. Suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum, vagina, or urethra. After insertion, suppositories soften, melt or dissolve in the cavity fluids. In general, for suppositories, traditional carriers may include, for example, polyalkylene glycols, triglycerides, or combinations thereof. In certain embodiments, suppositories may be formed from mixtures containing, for example, the active ingredient in the range of about 0.5% to about 10%, such as about 1% to about 2%.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and/or the other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsion, certain methods of preparation may include vacuum-drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered liquid medium thereof. The liquid medium should be suitably buffered if necessary and the liquid diluent first rendered isotonic prior to injection with sufficient saline or glucose. The preparation of highly concentrated compositions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small area.

The composition must be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less that 0.5 ng/mg protein.

In particular embodiments, prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin, or combinations thereof. 2. Combination Therapy

In order to enhance or increase the effectiveness of a bisphenol of the present invention, the bisphenol may be combined with, e.g., another anticancer agent or method of anticancer therapy, such as surgery or radiation. A bisphenol may also be combined with another drug in order to mitigate toxic side effects associated with that drug. A non- limiting example of a side effect associated with topoisomerase II poisons is weight loss. It is contemplated that this type of combination therapy may be used in vitro or in vivo. In a non-limiting example, an anticancer agent may be used in combination with a bisphenol. A non-limiting example of an anticancer agent is a topoisomerase II poison. Another non-limiting example of an anticancer agent is radiation.

For example, bisphenols of the present invention may be provided in a combined amount with an effective amount of an anticancer agent to reduce or block DNA replication in cancerous cells (e.g., tissues, tumors). A bisphenol of the present invention may also be combined with a topoisomerase II poison, wherein the bisphenol acts to protect tissues from toxic effects associated with the poison. These processes may involve administering the agents at the same time or within a period of time wherein separate administration of the substances produces a desired therapeutic benefit. This may be achieved by contacting the cell, tissue, or organism with a single composition or pharmacological formulation that includes two or more agents, or by contacting the cell with two or more distinct compositions or formulations, wherein one composition includes one agent and the other includes another.

The compounds of the present invention may precede, be co-current with and/or follow the other agents by intervals ranging from minutes to weeks. In embodiments where the agents are applied separately to a cell, tissue or organism, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agents would still be able to exert an advantageously combined effect on the cell, tissue or organism. For example, in such instances, it is contemplated that one may contact the cell, tissue or organism with two, three, four or more modalities substantially simultaneously (i.e., within less than about a minute) as the candidate substance. In other aspects, one or more agents may be administered about 1 minute, about 5 minutes, about 10 minutes, about 20 minutes about 30 minutes, about 45 minutes, about 60 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours, about 48 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 1, about 2, about 3, about 4, about 5, about 6, about 7 or about 8 weeks or more, and any range derivable therein, prior to and/or after administering the candidate substance.

Examples of how one may determine the timing of administration of a bisphenol, a topoisomerase II poison (e.g., etoposide), and/or radiation are set forth in U.S. Patent No. 6,265,385 and U.S. Publ. Appl. No. 2007/0185124, each of which are incorporated by reference in its entirety.

Various combination regimens of the agents may be employed. Non-limiting examples of such combinations are shown below, wherein a bisphenol is "A" and a second agent, such as an anticancer agent, is "B":

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A E. Examples

The following examples are included to demonstrate certain preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. Abbreviations: 3D-QSAR, three-dimensional quantitative structure-activity relationship; CHO, Chinese hamster ovary cells; CoMFA, comparative molecular field analysis; CoMSIA, comparative molecular similarity index analysis; /C₅₀, 50% inhibitory concentration; kDNA, kinetoplast DNA; pICso, -log (/C₅₀) (in molar concentration units); MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; MTT, 3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Na₂EDTA, disodium ethylenediaminetetraacetic acid. pBR322 plasmid DNA was obtained from MBI Fermentas (Burlington, Canada) and the kDNA from TopoGEN (Columbus, OH). HindIII was from Invitrogen (Burlington, Canada). Unless indicated, other chemicals were from Sigma- Aldrich (Oakville, Canada). The MTS CellTiter 96® AQueous One Solution Cell Proliferation Assay kit was obtained from Promega (San Luis Obispo, CA). The structures of the compounds tested are shown in FIG. 1. DHPDPSl, DHPDS2, SCHOl, SCHO2, OCHOl, OCHO2, S3OH, S4OH, O3OH, bisAC3, bispCP5 and cyclofenil diphenol were synthesized and characterized by ¹H- and ¹³C-nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry as described below. DHDP, TDP, EIBP, BHPM, 4,4'-dihydroxybenzophenone and EBP were purchased from Sigma-Aldrich (Oakville, Canada) and the NSC-labeled compounds were obtained from the National Cancer Institute (Bethesda, MD). The linear and non- linear least squares analyses were done with SigmaStat (Systat, Point Richmond, CA).

All molecular modeling was done using SYBYL 7.2.3 (Tripos, St. Louis, MO) on a Hewlett-Packard XW4100 PC workstation with a Redhat Enterprise 3 Linux operating system. All molecules were built using SYBYL. Geometry-optimization was carried out with the Tripos force field using a conjugate gradient with a convergence criterion of 0.01 kcal/mol and Gasteiger-Huckel charges and a distance- dependent dielectric constant.

Melting points were taken on a Gallenkamp melting point apparatus and are uncorrected. Electrospray ionization mass spectra (ESI-MS) were acquired on a Quattro-LC instrument (Micromass-Waters, Mississauga, Canada). Samples (~10^~3 M in acetonitrile) were injected in the Quattro ion source and electrosprayed at 4 kV with a carrying phase of 50:50 acetonitrile-water, flow rate 10 μL/min. Nitrogen gas was used as the sheath gas to facilitate the spraying. The cone (or declustering) voltage was adjusted at 20 V (positive ion mode) and 30 V (negative ion mode). Additional parameters included: source temperature (110⁰C) and nebulizer temperature (130⁰C). ¹H- and ¹³C-nuclear magnetic resonance (NMR) spectra were recorded at 300 K in 5 mm NMR tubes on Bruker (Milton, Canada) Avance 300 spectrometer operating at 300.13 MHz for ¹H-NMR and 75.5 MHz for ¹³C-NMR, respectively, on solutions in acetone -dβ, unless otherwise indicated. Chemical shifts are given in parts per million (ppm) (+/-0.01 ppm) relative to that of tetramethylsilane (TMS) (0.00 ppm) in the case of the ¹H-NMR spectra, and to the central line of chloroform-ύf (δ 77.2) or acetone-^ (δ 29.9) for the ¹³C-NMR spectra. TLC was performed on aluminum-backed plates bearing 200 μm silica gel 60 F254 (Silicycle, Quebec). Compounds were visualized by quenching of fluorescence by UV light (254 nm) where applicable and/or were located by putting the TLC plate in a iodine chamber until color developed. Compounds were purified on silica gel (Ultra pure grade, 230-400 mesh, Silicycle) by flash chromatography using the eluents specified. The ratios of the solvents used in TLC and column chromatography are volume ratios.

EXAMPLE 1

Synthesis of a bisphenol of the present invention: 4,4'-sulfonyl diphenol (DHDPSl) and 4,4'-dihydroxydiphenyl sulfoxide

(DHDPS2)

The following procedure represents a method of accessing DHDPSl and DHDPS2, although modifications to this method are possible, as recognized by a skilled artisan. These compounds may be accessed by other means as well, as known to a skilled artisan. Hydrogen peroxide (30 %, 160.8 mg, 1.42 mmol, 1.4 eq.) was added to a suspension of 4,4'-thiodiphenol ether (228.8 mg, 1.05 mmol), silica gel (200.0 mg, Silicycle, 230 - 400 mesh) and acetic anhydride (135.0 mg, 1.15 mmol, 1.1 eq.) in dichloromethane-methanol (5 ml, 4:1). The mixture was stirred at room temperature for 64 h and purified by column chromatography (ethyl acetate: hexanes 1.5: 1). Two colorless solid products were obtained: 4,4'-sulfonyl diphenol: Rf = 0.38, yield 30.0 mg (11.4 %); mp: 245.0-248.0⁰C (lit. (Zaccaro et al, 2002) 247°C); ¹H-NMR δ (DMSO-J6:CDC1₃ 2:1) 10.35 (s, 2H, OH), 7.65, 6.85 (dd, 4H, PhH); ¹³C-NMR δ (DMSO-J6:CDC1₃ 2:1) 161.5, 132.1 (tertiary PhC), 129.1, 115.8 (PhC) and 4,4'- dihydroxydiphenyl sulfoxide: Rf = 0.18; yield 185.1 mg (0.79 mmol, 75.2 %); mp: 198.0-200.0⁰C (lit. (Engman et al, 1994) 201⁰C); ¹H-NMR δ (DMSO-^:CDC1₃ 2:1) 9.93 (s, 2H, OH), 7.37, 6.84 (dd, 4H, PhH); ¹³C-NMR δ (DMSO-J6:CDC1₃ 2:1) 159.9, 1135.3 (tertiary PhC), 126.4, 115.9 (PhC); MS (ESI, m/z, %): Calcd. for [M- H]^" 233.0, Found: 233.3 (100).

EXAMPLE 2

Synthesis of a bisphenol of the present invention:

Bis(3-formyl-4-hydroxyphenyl) sulfide (SCHOl) and 2-hydroxy-5-(4- hydroxyphenylthio)benzaldehyde (SCH02)

The following procedure represents a method of accessing SCHOl and SCHO2, although modifications to this method are possible, as recognized by a skilled artisan. These compounds may be accessed by other means as well, as known to a skilled artisan.

A colorless suspension of 4,4'-thiodiphenol ether (0.60 g, 2.75 mmol) in dry toluene-acetonitrile (11 ml, 10:1 v/v) was treated with tin chloride (63 ml, 0.55 mmol) and tributylamine (509 ml, 2.20 mmol) and was stirred under argon atmosphere for 20 min at room temperature. Paraformaldehyde (0.38 g, 12.1 mmol) was added and the reaction mixture was stirred at 100⁰C for 8 h, poured into distilled water (100 ml), acidified with hydrochloric acid (IM) to pH 2 and extracted with ethyl acetate (3 x 20 ml). The combined organic layers were washed with saturated sodium bicarbonate aqueous solution (2 x 10 ml) and distilled water (10 ml), dried with anhydrous magnesium sulfate, and filtered to give a yellow solution. Purification by column chromatography (ethyl acetate: hexanes 1 : 4) yielded two products. Bis(3-formyl-4-hydroxyphenyl) sulfide (SCHOl) yellow crystals were obtained after recrystallization from acetone-hexanes-ethanol. Rf = 0.65 (ethyl acetate: hexanes 1 :2); yield 150.0 mg (20 %); mp: 160.5 - 161.0⁰C (lit. (Keum et al, 2004) 157°C); ¹H-NMR δ 10.97 (bs, 2H, OH), 10.01 (s, 2H, CHO), 7.85 (d, 2H, J = 2.4 Hz, PhH-2), 7.61 (dd, 2H, J = 2.4 Hz, J = 8.7 Hz, PhH-6), 7.00 (d, 2H, J = 8.7 Hz, PhH-5); ¹³C-NMR δ 197.1 (C=O), 158.6, 140.6, 137.1, 126.7, 122.3, 119.2 (PhC); MS (ESI, m/z, %): Calcd. for [M-H]^" 273.03, Found: 273.3 (100).

2-hydroxy-5-(4-hydroxyphenylthio)benzaldehyde (SCHO2) as a yellow solid was obtained after chromatography Rf = 0.45 (ethyl acetate : hexanes = 1 : 2), yield 220.0 mg (33 %); mp: 108.50-109.5⁰C; ¹H-NMR δ 10.90, 8.81 (2 x bs, 2H, 2 x OH), 9.99 (s, IH, CHO), 7.70 (d, IH, J = 2.4 Hz, PhH-2), 7.48 (dd, IH, Ji = 2.4 Hz, J₂ = 8.7 Hz, PhH-6), 7.29 (m, 2H, PhH), 6.95 (d, IH, J = 8.7 Hz, PhH-5), 6.85 (m, 2H, PhH); ¹³C-NMR δ 197.2 (C=O), 158.3, 139.3, 135.5, 135.4, 128.9, 124.5, 122.1, 118.9, 117.1 (PhC); MS (ESI, m/z, %): Calcd. for [M-H]^" 245.0, Found: 245.2 (100).

EXAMPLE 3

Synthesis of a bisphenol of the present invention:

5-(3-formyl-4-hydroxyphenoxy)-2-hydroxybenzaldehyde (OCHO2) and 2- hydroxy-5-(4-hydroxyphenoxy)benzaldehyde (OCHOl)

The following procedure represents a method of accessing OCHO2 and OCHOl, although modifications to this method are possible, as recognized by a skilled artisan. These compounds may be accessed by other means as well, as known to a skilled artisan.

Using similar procedures to those described above for SCHOl and SCHO2 (Example 2), OCHO2 and OCHOl were obtained from 4,4'-dihydroxydiphenyl ether (520.0 mg, 2.57 mmol). 5-(3-Formyl-4-hydroxyphenoxy)-2-hydroxybenzaldehyde (OCHO2) was obtained as a yellow solid after chromatography; Rf = 0.41 (ethyl acetate: hexanes = 1 : 3); yield 30.0 mg (5 %); mp 118.5 - 120.0⁰C; ¹H-NMR δ 10.58 (bs, 2H, 2 x OH), 9.87 (s, 2H, CHO), 7.30 (d, 2H, J = 3.0 Hz), 7.23 (dd, 2H, Ji = 3.0 Hz, J₂ = 8.9 Hz), 6.90 (d, IH, J = 8.9 Hz); ¹³C-NMR δ 196.9 (C=O), 157.9, 150.7, 121.7 (tertiary PhC), 128.8, 122.4, 119.4 (PhC); MS (ESI, m/z, %): Calcd. for [M-H]^" 257.1, Found: 257.3 (100). 2-Hydroxy-5-(4-hydroxyphenoxy)benzaldehyde (OCHOl)) was obtained as a yellow solid after chromatography; Rf = 0.31 (ethyl acetate: hexanes = 1 : 3), yield 170.0 mg (29 %); mp: 122.0 - 124.0⁰C; ¹H-NMR δ 10.66, 8.28 (2 x bs, 2H, 2 x OH), 9.98 (s, IH, CHO), 7.30 (d, IH, J = 3.0 Hz), 7.25 (dd, IH, Ji = 3.0 Hz, J₂ = 9.0 Hz), 6.97 (d, IH, J = 9.0 Hz), 6.99 - 6.82 (m, 4H); ¹³C- NMR δ 197.0 (C=O), 154.3, 151.9, 150.3, 121.6 (tertiary PhC), 128.2, 121.2, 120.7, 119.0, 116.8 (PhC); MS (ESI, m/z, %): Calcd. For [M-H]^" 229.1, Found: 229.3 (100).

EXAMPLE 4 Synthesis of a bisphenol of the present invention:

3,4,4'-trihydroxydiphenyl sulfoxide (S3OH)

The following procedure represents a method of accessing S3OH, although modifications to this method are possible, as recognized by a skilled artisan. This compound may be accessed by other means as well, as known to a skilled artisan. A solution of hydrogen peroxide (30 %, 166 μl) in water (994 μl) was added to a solution of SCHO2 (0.18 g, 0.73 mmol) in aqueous sodium hydroxide (2 %, 3.5 ml) dropwise. The obtained orange solution was stirred for 2.5 h in an argon atmosphere. The obtained orange-red solution was acidified by hydrochloric acid (1 M, 3 ml) and extracted with ethyl acetate (4 x 3 ml). The combined organic layers were washed with distilled water (3 ml), dried (anhydrous magnesium sulfate), filtered and concentrated to an orange-red syrup. Purification by flash column chromatography (ethyl acetate: hexanes = 3: 2) yielded the title compound as an orange syrup. Rf 1.2 (ethyl acetate: hexanes = 2: 1); yield 90.0 mg (49 %); IH-NMR δ 8.80 (bs, 3H; 3 x OH), 7.49 (ddd, Ji = 2.0 Hz, J₂ = 2.8 Hz, J3 = 8.7 Hz, 2H,), 7.14 (d, J = 2.1 Hz, IH), 7.03 (dd, Ji = 2.1 Hz, J₂ = 8.2 Hz, IH), 6.95 (ddd, Ji = 2.0 Hz, J₂ = 2.8 Hz, J3 = 8.7 Hz, 2H), 6.93 (d, J = 8.2 Hz, IH); 13C-NMR 8160.7, 148.8, 146.6, 136.8, 136.4, 127.4, 118.0, 116.7, 116.2, 112.0; MS (ESI, m/z, %): Calcd. for [M-H]^" 249.0, Found: 249.2 (100).

EXAMPLE 5 Synthesis of a bisphenol of the present invention:

3,3',4,4'-tetrahydroxydiphenyl sulfoxide (S4OH) and 4-(4- hydroxyphenoxy)benzene-l,2-diol (030H)

The following procedure represents a method of accessing S4OH and O3OH, although modifications to this method are possible, as recognized by a skilled artisan. These compounds may be accessed by other means as well, as known to a skilled artisan.

Using a similar procedure as described for the preparation of S3OH (Example 4), S4OH and O3OH were obtained from SCHOl (90.0 mg, 0.33 mmol) and OCHOl (53.4 mg, 0.23 mmol), respectively. S40H was obtained as a yellow syrup. Rf 0.17 (ethyl acetate); yield 35.0 mg (40 %); ¹H-NMR δ 8.53 (bs, 4H; 4 x OH), 7.09 (d, J = 2.0 Hz, 2H, H-2), 7.01 (dd, J₁ = 2.0 Hz, J₂ = 8.2 Hz, 2H, H-6), 6.91 (d, J = 8.2 Hz, IH, H-5); ¹³C-NMR δ 148.5, 146.4, 137.7, 117.8, 116.1, 111.8; MS (ESI, m/z, %): Calcd. for [M-H]^" 265.0, Found: 265.2 (100). O3OH was obtained as a colorless solid. Rf 0.20 (ethyl acetate: hexanes = 3: 2); yield 15.0 mg, (30 %); mp 129.5-131.0⁰C; ¹H- NMR δ 7.63 (s, 3H), 6.65 (s, 7H); ¹³C-NMR 8150.9, 150.8, 116.3, 116.2; MS (ESI, m/z, %): Calcd. for [M-H]^" 217.1, Found: 217.2 (100).

EXAMPLE 6 Synthesis of bisphenols of the present invention:

McMurry coupling reactions

McMurry coupling was used to access certain bisphenols of the present invention. While the following represents a McMurry coupling protocol as described by Seo et al, 2006, persons of skill in the art recognize that modifications to this protocol are possible and that other means of performing McMurry couplings are possible.

Titanium (VI) chloride (370 ml, 0.82 g, 4.31 mmol) was added dropwise to a grey suspension of zinc powder (0.60 g, 9.00 mmol) in dry THF (10 ml) at -18°C (a sodium chloride-ice bath). Yellow fumes were released during the addition. The obtained yellow-green mixture was refluxed for 2 h at 100⁰C, cooled to room temperature, and treated with a solution of 4,4'-dihydroxybenzophenone (0.25 g, 1.16 mmol) and with each of the ketones (acetone, cyclohexanone, or cyclopentanone, respectively) (1.16 mmol) in THF (8 ml) in separate reactions. After refluxing for 2 h, the mixture was cooled to room temperature and slowly poured into a saturated aqueous sodium bicarbonate solution (100 ml). Ether (100 ml) was then added with vigorous stirring. The mixture was filtered through Celite and extracted with ether (2 x 50 ml). The combined organic phases were dried over anhydrous magnesium sulfate, filtered and concentrated to yield a light yellow residue. Purification by flash column chromatography (ethyl acetate: hexanes = 1 : 3) yielded each coupling product.

4-(l-(4-Hydroxyphenyl)-2-methylprop-l-enyl)phenol (bispAC3). Yield 141 mg (51%); colorless needles; mp 181.5-183.0⁰C; ¹H-NMR (MeOD-^) δ 6.96 (dd, J₁ = 1.7 Hz, J₂ = 8.6 Hz, 4H), 6.70 (dd, J₁ = 1.7 Hz, J₂ = 8.6 Hz, 4H), 1.76 (s, 6H); ¹³C- NMR (MeOD-^)δ 156.4, 138.1, 136.6, 132.0, 131.0, 115.6, 33.9, 22.7; MS (ESI, m/z, %) calcd. For C₁₆H_nO₂ [M+H]⁺ 241.1. Found: 241.1 (100).

Bis(4-hydroxyphenyl)methylenecyclopentane (bispCPS). Yield 131 mg (42 %); a colorless solid; mp 193.0-195.0⁰C (ref:(Seo et al, 2006) 194-195°C); ¹H-NMR (MeOD-^) δ 6.96 (dd, Ji = 1.5 Hz, J₂ = 8.6 Hz, 4H), 6.70 (dd, Ji = 1.5 Hz, J₂ = 8.6 Hz, 4H), 2.36 (m, 4H), 1.67 (m, 4H); ¹³C-NMR (MeOD-^) δ 156.5, 142.0, 136.7, 134.2, 131.3, 115.6, 33.9, 27.9; MS (ESI, m/z, %) calcd. For Ci₈H_nO₂ [M-H]^" 265.1, Ci₈H_nO [M-OH]^" 249.1. Found: 265.2 (10), 249.1 (100).

Bis(4-hydroxyphenyl)methylenecyclohexane (cyclofenil diphenol). Yield 55 mg (17%); a colorless solid; mp 230.0-233.0⁰C (ref:(Seo et al, 2006) 235-237°C); ¹H-NMR (MeOD-c#) δ 6.88 (dd, Ji = 2.3 Hz, J₂ = 8.6 Hz, 4H), 6.88 (dd, Ji = 2.1 Hz, J₂ = 8.6 Hz, 4H), 2.21 (bs, 4H), 1.57 (bs, 6H); ¹³C-NMR (MeOD-^) δ 156.3, 138.7, 136.4, 135.5, 131.9, 115.7, 33.5, 29.8, 28.0; MS (ESI, m/z, %) calcd. For Ci₉H₂₀O₂Na [M+Na]⁺ 303.2. Found: 303.0 (100).

EXAMPLE 7

Cell culture and growth inhibition studies

These studies were performed to investigate cancerous cell growth inhibition by certain bisphenols of the present invention. Human leukemia K562 cells, obtained from the American Type Culture Collection and K/VP.5 cells (a 26-fold etoposide- resistant K562-derived sub-line with decreased levels of topoisomerase Ilα mRNA and protein) (Fattman et al, 1996) were maintained as suspension cultures in DMEM (Dulbecco's Modified Eagle Medium, Invitrogen, Burlington, Canada) containing 10% fetal calf serum and 2 mM L-glutamine. The spectrophotometric 96-well plate cell growth inhibition MTS assay, which measures the ability of the cells to enzymatically reduce MTS after drug treatment, has been described (Liang et al, 2006). The drugs were dissolved in dimethyl sulfoxide. The final concentration of dimethyl sulfoxide did not exceed 0.5% (v/v) and was an amount that had no detectable effect on cell growth. The cells were incubated with the drugs for the times indicated and then assayed with MTS. IC ₅₀ values for growth inhibition in both assays were measured by fitting the absorbance-drug concentration data to a four-parameter logistic equation as described (Liang et al, 2006). CHO cells (type AA8; ATCC CRL-1859), obtained from the American Type Culture Collection (Rockville, MD) and DZR cells (a dexrazoxane-resistant CHO cell line previously described) (Hasinoff et al., 1997; Hasinoff and Wu, 2003) were grown in alpha minimum essential medium (α-MEM; Invitrogen, Burlington, Canada) containing 20 mM HEPES (4-(2- hydroxyethyl)piperazine-l-ethanesulfonic acid; Sigma, St. Louis, MO)) and assayed for their growth inhibitory effects using the MTT assay as described (Hasinoff et al, 2004).

Results: Effect of the bisphenols on K562 cell growth inhibition. Examples of the growth inhibitory effects of the bisphenols S4OH and DHDP on K562 cells are shown in FIG. 2A. The IC^ data for the growth inhibitory effects of all the bisphenols tested on K562 and KTVP.5 cells are given in Table 1. It can be seen from the data in Table 1 from a comparison of the structure of DHDP with the 4,4'-bisphenol analogs TDP, BHPM, EIPB, DHDS2 and DHDSl that an electronegative atom such as a S or O in the bridge between the phenyl rings increased growth inhibitory activity compared to a CH₂ or CH functionality and also that extension of the bridge to a CH₂CH₂ also decreased activity. Further substitution (as in bisphenol A) or conversion of the methyl substituted carbon linker in EIPB into either a ketone (4,4'- dihydroxybenzophenone) or double bonded conjugates (bispAC3, bispCP5, or bispCHβ (also called cyclofenil diphenol)) had little positive effect on the activity. Conversion of the S to a sulfone or sulfoxide also decreased activity. A comparison of the sulfones S4OH, DHDPS2 and S3OH indicated that electron donor groups at the 3,3' positions increased activity.

The effect of the bisphenols on the growth of a K562 cell line compared to the K/VP.5 cell line with a decreased level of topoisomerase Ha. One method by which cancer cells increase their resistance to topoisomerase II poisons is by lowering their level or activity of topoisomerase II (Fortune and Osheroff, 2000; Ritke et al, 1994). With less topoisomerase II in the cell, cells produce fewer DNA strand breaks and topoisomerase II poisons are less lethal to cells. These cell lines provide a convenient way to test whether a drug that inhibits topoisomerase II acts as a topoisomerase II poison (Hasinoff et al., 2007; Hasinoff et al., 2005). Conversely, a lack of change in sensitivity of a putative topoisomerase II poison to a cell line with a lowered topoisomerase II level can be taken to indicate that poisoning of topoisomerase II was not an important mechanism for this particular agent. The KTVP.5 cell line with acquired resistance to etoposide contains one-fifth the topoisomerase Ilα content of the parental K562 cells (Fattman et al., 1996). The IC^ for growth inhibition of K562 cells and KTVP.5 cells, as measured with the MTS assay, after a 72 h continuous treatment with a range of bisphenol concentrations are compared in Table 1. None of the bisphenols were very cross resistant, which again suggests that the bisphenols did not act as topoisomerase II poisons. Because the bisphenols display some structural resemblance to the bisdioxopiperazine dexrazoxane (ICRF-187) in that both have bis-substituted ring systems joined by linker atoms, the ability of the bisphenols to inhibit the growth of the dexrazoxane-resistant DZR cell line was determined (Hasinoff et al, 1997; Hasinoff and Wu, 2003; Hasinoff et al, 2004). The DZR cell line, which was derived from the parent CHO cell line and has a Thr48Ile mutation in topoisomerase II, is 400-fold resistant to dexrazoxane (Hasinoff et al, 1997; Hasinoff and Wu, 2003; Hasinoff et al., 2004). This mutation is located in the N-terminal ATP binding region of topoisomerase II close to the dexrazoxane binding site (Classen et al., 2003a; Classen et al., 2003b) and likely interferes with dexrazoxane binding. The results in Table 1 show that none of the bisphenols were very cross resistant to the DZR cell line, which indicates that the bisphenols did not inhibit the catalytic activity of topoisomerase Ilα by binding to the bisdioxopiperazine binding site.

The effect of bisphenols on the K562 cell growth inhibitory effects of the topoisomerase II poisons doxorubicin and etoposide. The present inventors and others have shown that the bisdioxopiperazine topoisomerase II catalytic inhibitors dexrazoxane and ICRF- 193 antagonized the growth inhibitory effects of doxorubicin, daunorubicin, etoposide and amsacrine (Hasinoff et al, 1996; Ishida et al, 1991; Sehested et al, 1993; Tanabe et al, 1991). Likewise, the purine NSC35866 (Jensen et al, 2005) can antagonize etoposide-induced growth inhibitory effects. In the case of dexrazoxane it may do this by trapping the enzyme in the form of a closed protein clamp, thus preventing the formation or stabilization of the topoisomerase II-DNA intermediate (Ishida et al, 1991; Sehested et al, 1993; Tanabe et al, 1991).

In order to determine if the bisphenols acted similarly to dexrazoxane (Hasinoff et al, 1996) and NSC35866 (Jensen et al, 2005) in antagonizing doxorubicin or etoposide growth inhibitory effects on K562 cells, experiments were carried out in which K562 cells were pretreated with either 5 μM O3OH or S4OH for 30 min prior to treatment with doxorubicin (FIG. 5A and 5B) and then continuously incubated with both drugs for 72 h prior to the MTS assay. These concentrations of O3OH or S4OH are about 6-fold higher than that required to inhibit the catalytic activity of topoisomerase Ilα (Table 1), but were not high enough to significantly inhibit the growth of the K562 cells. As shown in FIG. 5 A and 5B neither O3OH nor S4OH had much effect on the doxorubicin /C50 value. O3OH increased the doxorubicin /C50 value from 0.065 to 0.14 μM, while S4OH decreased it 0.017 μM. Thus, it can be concluded that neither of these bisphenols antagonized doxorubicin growth inhibition of K562 cells through their ability to inhibit topoisomerase Ilα.

The ability of 300 μM of the weaker topoisomerase Ilα inhibitor DHDP and S4OH to antagonize etoposide-induced growth inhibition of K562 cells were also evaluated. A 30 min pretreatment with the bisphenol was followed by a 1 h treatment with etoposide, after which both drugs were washed off. The concentrations of DHDP and S4OH were sufficiently high (10- and 400-fold, respectively) to strongly inhibit the catalytic activity of topoisomerase Ilα (Table 1). As shown in FIG. 5C DHDP increased the etoposide /C₅₀ nearly 6-fold from 17 to 97 μM, indicating that it antagonized the growth inhibitory effects of etoposide. However, as shown in FIG. 5D, S4OH, as with the doxorubicin treatment, had little effect, as it decreased the /C50 only slightly to 12 μM. A similar attempt was made to evaluate O3OH (30 μM) and SCHOl (200 μM). However, these compounds could not be evaluated, as even after washing both these drugs were growth inhibitory (data not shown) likely due to retained drug

EXAMPLE 8

Topoisomerase Ilα kDNA decatenation inhibition assay

Assays were performed to assess the ability of certain bisphenols of the present invention to inhibit topoisomerase Ilα decatenation inhibition. A spectrofluorometric decatenation assay was used to determine the inhibition of topoisomerase Ilα by the bisphenols (Hasinoff et ah, 2005; Hasinoff et ah, 2004; Liang et ah, 2006). kDNA consists of highly catenated networks of circular DNA. Topoisomerase Ilα decatenates kDNA in an ATP-dependent reaction to yield individual minicircles of DNA. The 20 μl reaction mixture contained 0.5 mM ATP, 50 mM Tris-HCl (pH 8.0), 120 mM KCl, 10 mM MgCl₂, 30 μg/ml bovine serum albumin, 50 ng kDNA, test compound (0.5 μl in dimethyl sulfoxide) and 20 ng of topoisomerase Ilα protein (the amount that gave approximately 80% decatenation). Using a high copy yeast expression vector, full-length human topoisomerase Ilα was expressed, extracted and purified as described previously (Hasinoff et ah, 2005). The final dimethyl sulfoxide concentration of 2.5% (v/v) was shown in controls not to affect the activity of topoisomerase Ilα. The assay incubation was carried out at 37°C for 20 min and was terminated by the addition of 12 μl of 250 mM Na₂EDTA. Samples were centrifuged at 8000 g at 25°C for 15 min and 20 μl of the supernatant was added to 180 μl of 600-fold diluted PicoGreen dye (Molecular Probes, Eugene, OR) in a 96-well plate. The fluorescence, which was proportional to the amount of kDNA, was measured in a Fluostar Galaxy (BMG, Durham, NC) fluorescence plate reader using an excitation wavelength of 485 nm and an emission wavelength of 520 nm. Results: The bisphenols inhibit the decatenation activity of topoisomerase

Ua. As shown in FIG. 2B for S4OH and DHDP and in Table 1 the bisphenols inhibited the decatenation activity of human topoisomerase Ilα. This assay is a measure of the ability of these compounds to inhibit the catalytic activity only and is not a measure of whether these compounds acted as topoisomerase II poisons as do some widely used anticancer drugs (Fortune and Osheroff, 2000; Li and Liu, 2001). The activity of the sulfoxide S4OH was close in activity to the O ether O3OH. An electronegative atom such as a sulfoxide (as in S4OH) or a O (as in O3OH) or S (as in SCHOl and SCHO2) in the bridge between the phenyl rings tends to increase topoisomerase Ilα inhibitory activity compared to a CH₂ or a CH₂CH₂ bridge, although such alkyl substituents are still active. Substitution of the 3,3' position in the O ether DHDP with a single OH (as in O3OH) increased activity while replacement with one or two formyl groups did not greatly affect activity. The most potent topoisomerase Ilα inhibitor, the biscatechol sulfoxide S4OH, was also more potent than S3OH, indicating a preference for 3,3' electronegative substitution. The two sulfones tested NSC38049 and NSC40763 were of lower activity suggesting a sulfoxide (or an S or O) was more favorable for activity. As with K562 growth inhibitory activity, further substitution or conversion of the linker in EIPB (as in bisphenol A, 4,4'-dihydroxybenzophenone, bispAC3, bispCP5, or bispCHβ (also called cyclofenil diphenol)) did not improve the activity. EXAMPLE 9

QSAR correlation analyses of CHO on K562 cell growth inhibition with topoisomerase Ilα inhibition

A QSAR correlation analysis was performed to analyze the relationship of cell growth inhibition demonstrated by certain bisphenols of the present invention with the topoisomerase Ilα decatenation inhibition activity of those bisphenols. As shown in

FIG. 3A and 3B the logarithms of the CHO and K562 /C50 data were significantly correlated with the logarithm of the /C₅₀ data for the catalytic inhibition of topoisomerase Ilα (r = 0.56, p < 0.001 and r = 0.24, p = 0.02, respectively). The significant correlation of the CHO and K562 /C50 data with topoisomerase Ilα /C50 suggests that inhibition of topoisomerase Ilα by the bisphenols contributed to the inhibition of cell growth

EXAMPLE 10 pBR322 DNA cleavage assays Several widely used anticancer agents, including doxorubicin and the other anthracyclines, mitoxantrone and etoposide, (Fortune and Osheroff, 2000; Li and Liu, 2001) are thought to be cytotoxic by virtue of their ability to stabilize a covalent topoisomerase II-DNA intermediate (the cleavable complex) and act as what are called topoisomerase II poisons. Thus, DNA cleavage assay experiments (Burden et al, 2001), as previously described (Hasinoff et al, 2006), were carried out using 250 μM etoposide as a control to see whether 250 μM of the test compounds stabilized the cleavable complex to produce linear DNA.

Topoisomerase Il-cleaved DNA complexes produced by anticancer drugs may be trapped by rapidly denaturing the complexed enzyme with sodium dodecyl sulfate (Burden et al, 2001; Liang et al, 2006). The drug-induced cleavage of double- stranded closed circular pBR322 DNA to form linear DNA was followed by separating the sodium dodecyl sulfate-treated reaction products using ethidium bromide gel electrophoresis as described (Burden et al, 2001; Liang et al, 2006). The 20 μl cleavage assay reaction mixture contained 100 μM of the drug, 150 ng of topoisomerase Ilα protein, 80 ng pBR322 plasmid DNA (MBI Fermentas, Burlington, Canada), 0.5 mM ATP in assay buffer (10 mM Tris-HCl, 50 mM KCl, 50 mM NaCl, 0.1 mM EDTA, 5 mM MgCl₂, 2.5% (v/v) glycerol, pH 8.0, and drug (0.5 μl in dimethyl sulfoxide). The order of addition was assay buffer, DNA, drug, and then topoisomerase Ilα. The reaction mixture was incubated at 37°C for 10 min and quenched with 1% (v/v) sodium dodecyl sulfate/25 mM Na₂EDTA. The reaction mixture was treated with 0.25 mg/ml proteinase K (Sigma) at 55°C for 30 min to digest the protein. The linear pBR322 DNA cleaved by topoisomerase Ilα was separated by electrophoresis (2 h at 8 V/cm) on a TAE (Tris base (4 mM)/glacial acetic acid (0.11% (v/v))/Na₂EDTA (2 mM) buffer)/ethidium bromide (0.5 μg/ml)/agarose gel (1.2%, wt/v)). Ethidium bromide was used in the gel and running buffer in order that the inhibition of relaxation activity could be measured along with formation of cleaved linear DNA. The DNA in the gel was imaged by its fluorescence on a Alpha Innotech (San Leandro, CA) Fluorochem 8900 imaging system equipped with a 365 nm UV illuminator and a CCD camera.

Results: Effect of bisphenols on the stabilization of the covalent topoisomerase Ha-DNA cleavable complex. As shown in FIG. 4A the addition of etoposide (lane 7) to the reaction mixture containing topoisomerase Ilα and supercoiled pBR322 DNA induced formation of linear DNA. Linear DNA was identified by comparison with linear pBR322 DNA produced by action of the restriction enzyme HindIII acting on a single site on pBR322 DNA (not shown). None of the bisphenols shown in FIG. 4A (DHDP, S4OH, O3OH, or OCHl) detectably increased formation of linear DNA. OCHOl, SCHOl, SCHO2, NSC402321, NSC40763, BHPM and EIPB were also tested and gave similar negative results. These results indicate that these bisphenols did not act as topoisomerase Ilα poisons.

As exemplified by dexrazoxane (Hasinoff et al., 1997), catalytic inhibitors of topoisomerase II may inhibit cleavable complex formation by topoisomerase II poisons such as etoposide (Andoh and Ishida, 1998; Jensen et al., 2006; Jensen et al., 2005; Larsen et al., 2003). Experiments were thus carried out to see if bisphenol pretreatment could antagonize etoposide-induced linear DNA formation by topoisomerase Ilα. As the results in FIG. 4B and 4C show, S4OH, O3OH, DHDP and SCHl all reduced the amount of linear DNA produced from etoposide-induced formation of the cleavable complex by amounts ranging from 29 to 84%.

EXAMPLE 11

Thermal denaturation of DNA assay

Assays were performed to determine whether certain bisphenols of the present invention bound to DNA. Compounds that intercalate into DNA stabilize the DNA double helix and increase the temperature at which the DNA is denatured (Priebe et al., 2001). The effect of 2 μM of the compounds on the change in the DNA thermal melt temperature (ΔT_m) of sonicated calf thymus DNA (5 μg/ml) was measured in 10 mM Tris-HCl buffer (pH 7.5) in a Cary 1 (Varian, Mississauga, Canada) double beam spectrophotometer by measuring the absorbance increase at 260 nm upon the application of a temperature ramp of l°C/min. The maximum of the first derivative of the absorbance-temperature curve was used to obtain the ΔT_m. Doxorubicin (2 μM), which is a strong DNA intercalator, was used as a positive control (Priebe et al., 2001). Results: Doxorubicin (2 μM), which is a well known DNA intercalating drug

(Priebe et al., 2001), was used as a control and was observed to increase the ΔT_m of sonicated DNA by 13.2° C from 71.0⁰C. The following compounds were tested at 2 μM to see if they affected the ΔT_m: NSC38049, NSC85582, NSC402321, DHDP, NSC58409, SCHOl, SCHO2, TDP, BHPM, and EIPB. None of these compounds significantly changed the ΔT_m and thus it can be concluded that they did not act, at least at the concentrations tested, by binding to DNA.

EXAMPLE 12 3D-QSAR analyses

Molecular modeling was performed to analyze which structural features of certain bisphenols of the present invention contributed to topoisomerase Ilα inhibition.

The CoMFA and CoMSIA analyses require that the 3D structures of the molecules be aligned to a core conformational template that is their presumed active form (Cramer III et al., 1988; Klebe et al., 1994; Kubinyi et al., 1998). For the CoMFA analysis, steric and electrostatic field energies were calculated using a sp³ carbon with a van der Waals radius of 1.52 A as the steric probe and a +1 charge as an electrostatic probe. Steric and electrostatic interactions were calculated using the Tripos force field with a distance-dependent dielectric constant at all lattice points of a regular spaced (2 A) grid. The energy cutoff was 30 kcal/mol. The alignment and lattice box used for the CoMFA calculation were also used to calculate similarity index fields for the CoMSIA analysis. Steric, electrostatic, hydrophobic, hydrogen bond donor and acceptor fields were evaluated in the CoMSIA analysis. Similarity indices were computed using a probe atom with +1 charge, radius 1 A, hydrophobicity +1, hydrogen bond donating +1, hydrogen bond acceptor +1, attenuation factor α 0.3 for the Gaussian-type distance. A partial least-squares (PLS) statistical approach, which is an extension of multiple regression analysis in which the original variables are replaced by a set of their linear combinations, was used to obtain the 3D-QSAR results. All models were investigated using the leave-one -out (LOO) method, which is a cross-validated partial least-squares method. The CoMFA and CoMSIA descriptors were used as independent variables and pICso was used as the dependent variable to derive the 3D-QSAR models. The q² (cross- validated correlation coefficient r²) and the optimum number of components (N) were obtained by the LOO method. The final model (non-cross-validated conventional analysis) was developed and yielded the non-cross-validated correlation coefficient r with the optimum number of components.

Results: 3D-QSAR modeling based on the inhibition of the decatenation activity of topoisomerase Ha and the growth inhibition of K562 cells. The wide range of topoisomerase Ilα inhibitory IC so values allowed us to carry out 3D-QSAR CoMFA and CoMSIA analyses in order to identify the structural features responsible for inhibitory activity. The energy-minimized structure of 0CH02 (FIG. 6A) was selected as the template molecule as it had meta and para substituents on both phenyl rings. The other compounds in the data set were prepared by modifying the structure of 0CH02 and energy minimizing all but the aromatic carbon atoms (FIG. 6A). The resulting aligned structures are shown in FIG. 6B. The results of the CoMFA and CoMSIA analyses of the ICso data for the inhibition of the topoisomerase Ilα are summarized in Table 2. Weakly inhibiting bisphenols with an /C50 larger than 570 μM were not included in the analyses due to inaccuracies in determining these values. No compound was found to be an outlier in the CoMFA analyses. However, S3OH was found to be an outlier in the CoMSIA analysis. Due to the relatively small numbers of compounds in each data set (19 and 18, respectively) all structures were used in the analyses, rather than dividing them into training and validation sets. The predicted and experimental pICso values for the CoMFA and CoMSIA analyses for inhibition of topoisomerase Ilα and K562 cell growth inhibition are plotted in FIG. 7A, 7B, 7C and 7C, respectively. The CoMSIA analysis was well correlated with an r value of 0.90 and a moderately good q² value of 0.46. However, the CoMFA analysis did not yield good r² or q² values. The CoMSIA analysis for the inhibition of the topoisomerase Ilα likely yielded a better model because, in addition to the steric and electrostatic contributions to the field, CoMSIA also measures hydrophobic and hydrogen bond donor and acceptor contributions to the field, and thus provides a more complete description of the interaction of the molecules with its binding site. The electrostatic contribution to the CoMSIA-derived field at 31.7% was the largest contributor to the overall field (Table 2). The hydrogen bond acceptor and hydrogen bond acceptor components at 25.2% and 23.7%, respectively, were the second and third largest contributors to the field (Table 2).

An examination of the four isocontour diagrams ("stdev*coeff, FIG. 6C, 6D, 6E and 6F) for the four largest field components for inhibition of topoisomerase Ilα that were mapped onto the OCHO2 molecule, shows the regions in space that were either favored or disfavored for inhibitory activity. The green contours indicate regions that increased inhibitory activity, and the red contours indicate regions that decreased inhibitory activity. An examination of the electrostatic field, which makes the largest contribution to the CoMSIA-derived field, showed that bisphenol analogs with polar substituents such as hydroxyl groups in the meta position on the phenyl rings were favored. For the hydrogen bond acceptor field, the second largest contributor to the CoMSIA-derived field, a meta-substituted hetero atom, as in an hydroxyl or formyl group was favored, while the sulfoxide or sulfone oxygens on the bridge were disfavored. For the hydrogen bond donor field para- and meta-substituted hydroxyl groups were favored. For the hydrophobic field, the region around the bridge atom was disfavored, which probably reflects the higher hydrophobicity of the alkyl substituents in this region.

CoMFA and CoMSIA analyses was also carried out on the /C50 data for K562 and K/VP.5 cell growth inhibition for the 23 bisphenols in Table 1. The CoMSIA analysis yielded q² values of 0.29 and 0.55, respectively, but with 6 and 7 optimum components respectively (Table 3). The r values of 0.91 and 0.97, respectively were quite good. The predicted and experimental pICso values for the CoMFA and CoMSIA analyses are plotted in FIG. 7C and FIG. 7D for the K562 cells. The electrostatic and hydrogen bond acceptor terms, respectively, made the largest contribution to the overall field, similar to what was found for the topoisomerase II CoMSIA analysis, as might be expected given that their activities are correlated (FIG. 3). As with the topoisomerase Ilα data, the CoMFA analysis did not yield high r² or q² values. All of the methods and apparatuses disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and apparatuses and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

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Claims

1. A method of inhibiting the catalytic decatenation activity of topoisomerase II in a cell, comprising administering to the cell an effective amount of a bisphenol, wherein the bisphenol is a compound of formula (I):

wherein:

D, G, J and L are each independently -H, a polar group, a hydrophobic group, a hydrogen bond donor, or a hydrogen bond acceptor; E and K are each independently a polar group, a hydrogen bond donor, or a hydrogen bond acceptor; and

P is a substituted or unsubstituted carbon or a substituted or unsubstituted heteroatom; provided that at least one of D, E and G is -OH and at least one of J, K and L is -OH.

2. The method of claim 1 , wherein topoisomerase II is topoisomerase Ilα.

3. The method of claim 1 , wherein:

D is a polar group, a hydrophobic group, a hydrogen bond donor, or a hydrogen bond acceptor;

G and L are each -H;

J is a polar group, a hydrogen bond donor, or a hydrogen bond acceptor; E and K are each independently a polar group, a hydrogen bond donor, or a hydrogen bond acceptor; and P is an unsubstituted heteroatom.

4. The method of claim 1, wherein any one or more polar groups or hydrogen bond donors of D, G, J, L, E, or K is a -OH group.

5. The method of claim 1, wherein the hydrogen bond acceptor of D, G, J, L, E, and/or K is a -OH or a -CHO group.

6. The method of claim 1, wherein P is -O- or -S-.

7. The method of claim 1, wherein any one of D, G, L, or J is a hydrogen bond acceptor.

8. The method of claim 1, wherein the hydrogen bond acceptor is further defined as a polar hydrogen bond acceptor.

9. The method of claim 1 , wherein the cell is in vitro.

10. The method of claim 1 , wherein the cell is in vivo.

11. The method of claim 1 , wherein the compound of formula (I) is further defined as a compound of formula (II):

wherein D, G, J, L and P are as defined in claim 1.

12. The method of claim 11, wherein D, G, J, L and P are further defined as the following:

D and L are each independently -H, -OH, -NO₂, or an alkyl group;

G and J are each independently -H, -OH, -CHO, an alkyl group, or -CH₂NR₅Re, wherein R₅ and R₆ are each independently an alkyl group; and P is -O-, -S-, -CO-, -SO-, -SO₂-, -CH₂-, -CR₇R₈, or -C=R₉Ri₀, wherein:

R₇ and R₈ are each independently -H or an alkyl group, provided that both are not H; and

R₉ and Rio are each independently an alkyl group or are joined together to form a cyclo alkyl group.

13. The method of claim 12, wherein the alkyl group of any one or more of D, L, G, J, R₅, Ke, R7, Rs, R9, or R₁O is each independently a lower alkyl group.

14. The method of claim 11, wherein P is -O-, -S-, or -SO-.

15. The method of claim 11 , wherein the compound of formula (II) is further defined as:

SCHO l 0CH02 03OH

16. The method of claim 15, wherein the compound of formula (I) is further defined as

17. The method of claim 1, wherein the compound of formula (I) is further defined as not any one or more of the following compounds:

DHDPSl BHPM

bisphenol A

EIBP 4,4-'dihydroxybenzophenone

18. The method of claim 1, wherein the bisphenol is comprised in a pharmaceutically acceptable composition.

19. A method of treating a patient with cancer, comprising administering to the patient an effective amount of a compound of formula (I):

wherein:

P is a substituted or unsubstituted carbon or heteroatom; provided that at least one of D, E and G is -OH and at least one of J, K and L is -OH.

20. The method of claim 19, wherein the cancer is cancer of the lung, liver, skin, eye, brain, gum, tongue, hematopoietic system or blood, head, neck, breast, pancreas, prostate, kidney, bone, testicles, ovary, cervix, gastrointestinal tract, lymph system, small intestine, colon, or bladder.

21. The method of claim 20, wherein the cancer is brain cancer.

22. The method of claim 20, further comprising administering a therapeutically effective amount of a topoisomerase II poison to the patient, wherein the therapeutically effective amount of the topoisomerase II poison is a cancer cell-killing amount and the therapeutically effective amount of the bisphenol is a topoisomerase II poison-protective amount.

23. The method of claim 19, wherein the cancer is not breast cancer or leukemia.

24. The method of claim 19, wherein the cancer is breast cancer and wherein the bisphenol is further defined as not any of the following:

DHDPSl BHPM

bisphenol A

EIBP 4,4-'dihydroxybenzophenone

25. A compound of formula (III) :

wherein: R₁₁ is -H or -OH; and R₁₂ is -CHO or -OH; provided that when R₁₁ is -OH, Y is -SO-, and when R₁₂ is -CHO, then Y is -O-.

26. The compound of claim 25, further comprised in a pharmaceutically acceptable composition.

27. A method of treating a patient with cancer, comprising administering to the patient an effective amount of a compound of claim 25.

28. The method of claim 27, further comprising administering a therapeutically effective amount of a topoisomerase II poison to the patient, wherein the therapeutically effective amount of the topoisomerase II poison is a cancer cell-killing amount and the therapeutically effective amount of the bisphenol is a topoisomerase II poison-protective amount.

29. A method of inhibiting the growth of a cell, comprising administering to the cell an effective amount of a compound of formula (I):

wherein:

30. The method of claim 29, wherein the cell is a cancer cell.

31. The method of claim 30, wherein the cancer cell is further defined as a lung cancer cell, liver cancer cell, skin cancer cell, eye cancer cell, brain cancer cell, gum cancer cell, tongue cancer cell, hematopoietic system or blood cancer cell, head cancer cell, neck cancer cell, breast cancer cell, pancreas cancer cell, prostate cancer cell, kidney cancer cell, bone cancer cell, testicles cancer cell, ovary cancer cell, cervix cancer cell, gastrointestinal tract cancer cell, lymph system cancer cell, small intestine cancer cell, colon cancer cell, or bladder cancer cell.

32. The method of claim 30, wherein the cancer cell is neither a breast cancer cell nor a leukemia cancer cell.

33. The method of claim 29, wherein the cell is in vitro.

34. The method of claim 29,wherein the cell is in vivo.