WO2012119010A9 - Materials for inhibiting aromatase and method of using the same to diagnose, treat and monitor breast cancer - Google Patents

Abstract

Disclosed herein are triphenylalkenes that can be used to inhibit aromatases. The inhibitory potency order of the tested compounds was as follows: norendoxifen » 4,4'-dihydroxy- tamoxifen > endoxifen > N-desmethyl-tamoxifen, N-desmethyl-4'-hydroxy-tamoxifen, tamoxifen-N-oxide, 4'-hydroxy-tamoxifen, N-desmethyl-droloxifene > 4-hydroxy-tamoxifen, tamoxifen. Norendoxifen inhibited recombinant aromatase via a competitive mechanism with a K _i of 35 nM. Norendoxifen inhibited placental aromatase with an IC₅₀ of 90 nM, while it inhibited human liver CYP2C9 and CYP3A with IC₅₀ values of 990 and 908 nM respectively. Inhibition of human liver CYP2C19 by norendoxifen appeared even weaker. No substantial inhibition of CYP2B6 and CYP2D6 by norendoxifen was observed. These compounds, and pharmaceutically acceptable formulations of these compounds, can be used to treat diseases, diagnose and monitory conditions, including some forms of breast cancer. Such compounds can be used alone or in combination with other therapies and therapeutic compounds for treating diseases such as cancer.

Description

MATERIALS FOR INHIBITING AROMATASE AND METHOD OP USING THE SAME TO DIAGNOSE, TREAT AND MONITOR BREAST CANCER

PRIORITY CLAIM

[0001] This application claims the benefit of United States provisional patent application number 61/488,152 filed on March 1, 2011, which is incorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENT RIGHTS

[0002] This invention was made with government support under W81XWH-1 1-1-0016 awarded by the Department of Defense. The government has certain rights in the invention.

FIELD OF THE INVENTION

(0003) This invention relates generally to producing and. using aromatase inhibitors to treat, diagnose and evaluate the progress of various diseases and conditions including breast cancer.

BACKGROUND

[0004] The aromatase pathway is the primary pathway for the production of estrogen in women after ovarian function subsides during the menopause or after another physiological change or medical intervention that reduces or eliminates ovarian function. For this reason, the enzyme aromatase is a target of many therapies that are designed to reduce estrogen in postmenopausal women, and thereby reduce their risk for recurrent breast cancer. The enzyme can also be valuably targeted as part of treatments for infertility in which reduction of systemic estrogen is used to stimulate pituitary function and increase ovarian function.

[0005] Unfortunately, therapy with the currently marketed aromatase inhibitors (AIs) results in marked side effects mat make the drugs difficult to tolerate for the 2-5 year period required for effective breast cancer treatment Toxicities are one of the major reasons for discontinuation of AIs, and the most common adherence-limiting toxicity is the AI-associatcd musculoskeletal syndrome, or AUvtSS. An object of the present invention is to provide benefit to patients who arc treated with aromatase inhibitors for breast cancer and other conditions by cxpaiHlmg their treatment options and enhancing the efficacy of their treatment.

[0006] Not all patients with aromatase inhibitors respond favourably. Accordingly, there is a need for a method of identifying patients that will benefit from this treatment with these molecules. Some embodiments of the invention include using aromatase inhibitor to diagnose and/or characterize a particular form of disease mat is characterized by aberrant aromatase activity. In some embodiments aromatase inhibitors, including the compoundsdisclosed herein, are used to track a given disease and/or to assess the effectiveness of a given therapy used to treat diseases such as breast cancer.

[0007] Still other embodiments include treating humans or animals with aromatase inhibitors such as those disclosed herein to treat diseases such as breast cancer.

SUMMARY

[0008] Some embodiments of the invention include at least one aromatase inhibitor having structures according to Formu

or a geometric isomer thereof, or a pharmaceutically acceptable salt or ester thereof, wherein,

the oxyalkylamine side chain on the phenyl group may have structural variants including: length in -(CH₂)„-, n = 1, 2, 3, 4 or 5, such as CH₂-CH₂-, CH₂-CH₂- CH₂-_, CH₂-CH₂-CH₂- CH₂- and CH₂-CH₂-CH₂-CH₂- CH₂- position on the phenyl group; position of oxygen in relation to - (CH₂)_n- chain; and oxyalkene composition of the side chain;

Ri may be independently selected from the group consisting of H, CH₃ and OH; R₂ and R₃ are independently selected from the group consisting of H, CH₃ ^", CH₃-CH₂-, CH₃-CH₂-CH₂-; R4 is selected from the group consisting of: H, CH₃ ^~, CH₃-(CH₂)_n-, hydoxy, methoxy, ethoxy; and n = 1, 2, 3, 4 or 5.

[0009] Still other embodiments of the invention include methods of inhibiting aromatase; comprising the step of: providing at least one compound according to Formula A

n = 2; Ri is independently selected from the group consisting of H, and OH; R₂ and R₃ are independently selected from the group consisting of H, or Me; and R4 is selected from the group consisting of: H, or Me.

[0010] In some embodiments the contacting step occurs in vitro, while in still other embodiments the contacting step occurs in vivo.

Some embodiments include, at least one aromatase inhibitor; according to Formula A;

or a geometric isomer thereof, or a pharmaceutically acceptable salt or ester thereof, wherein, the oxyalkylamine side chain on the phenyl group may have structural variants including: length in -(CH₂)_n-; position on the phenyl group; position of oxygen in relation to -(CH₂)_n- chain; and oxyalkene composition of the side chain; Ri may be independently selected from the group consisting of H, CH₃ and OH; R₂ and R₃ are independently selected from the group consisting of H, CH₃ ^", CH₃-CH₂-, CH₃-CH₂-CH₂-; R4 is selected from the group consisting of: H, CH₃ ^", CH₃- (CH₂)_n-, hydoxy, methoxy, ethoxy; and n = 1 , 2, 3, 4 or 5 such as CH₂-CH₂-, CH₂-CH₂- CH₂- CH₂-CH₂-CH₂- CH₂-and CH₂-CH₂-CH₂-CH₂- CH₂-.

[0011] In some embodiments the aromatase inhibitor has the same core as Formula A but is substituted with the following groups, n = 2; Ri is independently selected from the group

3 consisting of H, and OH; R₂ and R₃ are independently selected from the group consisting of H, or Me; and R4 is selected from the group consisting of: H, or Me.

[0012] Some embodiments include methods of inhibiting an aromatase; comprising the step of: providing at least one compound of Formula A, or an ester of pharmaceutically acceptable salt thereof; and contacting said compound with aromatase. In some embodiments the contacting step occurs in vitro. While in other embodiments, the contacting step occurs in vivo.

[0013] Some embodiments of the invention include methods of screening patients for treatment with aromatase inhibitors, comprising the steps of: providing at least one compound according to Formula A, measuring the level of aromatase activity in both the presence and in absence of said aromatase inhibitor in a sample of tissue, blood, cells and/or fluid from a patient; and attributing the change in aromatase activity measured in the presence and in the absence of said aromatase inhibitor to the level of aromatase in the sample that is sensitive to inhibition with at least one aromatase inhibitor. Some embodiments further include the step of comparing the level of aromatase activity in the sample that can be inhibited with the aromatase inhibitor with an amount of aromatase activity that is diagnostic for a given pathology. Some embodiments include the step of correlating the level of aromatase activity measured in the sample from the patient with the patient's likelihood of responding to a treatment regime.

[0014] In some embodiments the disease diagnosed, monitored or treated with at least one compound according to Formula A is breast cancer.

[0015] Some embodiments of the invention include methods of treating a patient, comprising the steps of: providing at least on

or a geometric isomer thereof, or a pharmaceutically acceptable salt or ester thereof, wherein, the oxyalkylamine side chain on the phenyl group may have structural variants including: length in - (CH₂)_n-; position on the phenyl group; position of oxygen in relation to -(CH₂)_n- chain; and oxyalkene composition of the side chain; Ri may be independently selected from the group consisting of H, CH₃ and OH; R₂ and R₃ are independently selected from the group consisting of H, CH₃ ^~, CH₃-CH₂-, CH₃-CH₂-CH₂-; R4 is selected from the group consisting of: H, CH₃ ^~, CH₃- (CH₂)_n-, hydoxy, methoxy, ethoxy; and n = 1, 2, 3, 4 or 5 such as .

[0016] In some embodiments the method treating a patient with at least one compound according to Formula A further includes the step of: administering at least a therapeutically effective dose of at least one compound according to formula A. In some embodiments the patient is either an animal or a human being, in some embodiments the patient is symptomatic for breast cancer. Some methods of treating a disease with at least once compound according to Formula A further include the step of monitoring the course of the diseasing by obtaining at least one more sample of tissue, cells, blood or fluid from the patient after said patient is treated with at least one therapeutic dose of the compound for Formula A.

[0017] In some embodiments a patient treated with at least one compound of Formula A is also treated with at least one other therapy selected from the group consisting of: radiation, convention chemotherapy and surgery.

[0018] Some embodiments of the invention include methods treating a patient, comprising the step: of providing a compound according to:

wherein, n = 2; Ri is independently selected from the group consisting of H, and OH; R₂ and R₃ are independently selected from the group consisting of H, or Me; and R4 is selected from the group consisting of: H, or Me, or an ester of pharmaceutically acceptable salt thereof. In some embodiments the treated patient is symptomatic for breast cancer. In some embodiments the patient is also treated with at least one other therapeutic compound. In some embodiments the patient is also treated with at least one other therapy selected from the group consisting of:

radiation therapy, convention chemotherapy and surgery.

Some aspects of the invention include inhibiting a aromatase; comprising the step of:

providinging at least one aromatase inhibitor of Formula A:

wherein, Ri may be independently selected from the group consisting of H, CH₃ and OH;

R₂ and R₃ are independently selected from the group consisting of H, CH₃ ^", CH₃-CH₂-, CH₃- CH₂-CH₂-; R₄ is selected from the group consisting of: H, CH₃ ^", CH₃-(CH₂)_n-, hydoxy, methoxy, ethoxy; and n = 1 , 2, 3, 4 or 5 and wherein Formula A inhibits at least one aromatase and contacting said at least one aromatase inhibitor with at least one aromatase.

[0019] In some embodiment the aromatase inhibitor of Formula A is one in which n = 2; Ri is independently selected from the group consisting of H, and OH; R₂ and R₃ are independently selected from the group consisting of H, or Me; and R₄ is selected from the group consisting of: H, or Me. In some preferred embodiments the aromatase inhibitor is selected from the group consisting of norendoxifen and endoxifen. In some embodiment the contacting step occurs in vitro while in some it occurs in vivo. In some preferred embodiments the aromatase inhibitor is a better inhibitor of aromatase CYP19 than it is an inhibitor of the aromatases selected from the group consisting of: CYP2B6, CYP2D6, CYP2C9, CYP2C19, and CYP3A.

[0020] Some aspects of the invention include methods of screening patients for treatment with aromatase inhibitors, comprising the steps of: contacting at least one aromatase inhibitor of Formula A:

wherein, Ri may be independently selected from the group consisting of H, CH₃ and OH; R₂ and R₃ are independently selected from the group consisting of H, CH₃ ^", CH₃-CH₂-, CH₃-CH₂-CH₂-; R4 is selected from the group consisting of: H, CH₃ ^~, CH₃-(CH₂)_n-, hydoxy, methoxy, ethoxy; and n = 1 , 2, 3, 4 or 5 and wherein the compound of Formula A inhibits at least one aromatase, with a sample, wherein said sample is a biological fluid or tissue; measuring the level of aromatase activity in both the presence and in absence of said aromatase inhibitor Formula A in a sample of tissue, blood, cells and/or fluid from a patient; and assigning a patient whose sample demonstrates a large change in aromatase activity measured in the presence and in the absence of said aromatase inhibitor to a group that is disposed to treatment with an aromatase inhibitor. In some preferred embodiments the the aromatase inhibitor is selected from the group consisting of endoxifen and norendoxifen or a pharmaceutically acceptable salt thereof.

[0021] Some aspects of the invention include methods of treating a patient, comprising the steps of: identifying a patient in need of an aromatase inhibitor; and administering a therapeutically effective amount of a compound according to Formula A, or a pharmaceutically acceptable salt thereof:

wherein, Ri may be independently selected from the group consisting of H, CH₃ and OH;

R₂ and R₃ are independently selected from the group consisting of H, CH₃ ^", CH₃-CH₂-, CH₃- CH₂-CH₂-; R4 is selected from the group consisting of: H, CH₃ ^", CH₃-(CH₂)_n-, hydoxy, methoxy, ethoxy; and n = 1 , 2, 3, 4 or 5 or a pharmaceutically acceptable salt thereof.

In some embodiments n = 2; Ri is independently selected from the group consisting of H, and OH; R₂ and R₃ are independently selected from the group consisting of H, or Me; and

R4 is selected from the group consisting of: H, or Me. Therapeutic doses are in the range of 10 to 40 mg per day, generally not to exceed more than 20 mg at a time. In some preferred embodiment the the aromatase inhibitor is endoxifen or a pharmaceutically acceptable salt thereof or norendoxifen or pharmaceutically acceptable salt thereof. In some embodiments the patient is an human or another mammal. In some embodiments the method of treating a patient further includes the step of: monitoring the course of the diseasing by obtaining at least one more sample of tissue, cells, blood or fluid from the patient after said patient is treated with at least one therapeutic dose of the compound for Formula A. In some preferred embodiment the patient treated with at least one aromatasc inhibitor of Formula A is also treated with at least one other compound and or with at least one other therapy.. These co-therapies can be selected from the group consisting of: radiation, convention chemotherapy and surgery. In some embodiments the treated patient is symptomatic for breast cancer. In some preferred embodiments the aromatase inhibitor is a better inhibitor of aromatasc CYP19 than it is an inhibitor of the aromatascs selected from the group consisting of: CYP2B6, CYP2D6, CYP2C9, CYP2C19, and CYP3A.

[0022] Some aspects of the invention include at least one aromatase inhibitor; comprising: an aromatase inhibitor of Formul

wherein, i may be independently selected from the group consisting of H, CHj and OH; R₂ and ¾ are independently selected from the group consisting of H, C¾~, CH3-CH4-, CH3-CH2-CH2-; R4 is selected from the group consisting of: H, CH₃', CHHCH-dn-, hydoxy, methoxy, ethoxy;

and n = 1, , 3, 4 or an w ?e ormu a nm > tS 8t castone In some embodiments of the invention n = 2; R₍ is independently selected from the group consisting of H, and OH; R2 and R₃ arc independently selected from fee group consisting of H, or Me; and R4 is selected from the group consisting of: H, or Me. In some preferred embodiments the aromatasc inhibitor is a better inhibitor of aromatase CYP1 man it is an inhibitor of the aromatascs selected from the group consisting of: CYP2B6, CYP2D6, CYP2C9, CYP2C19, and CYP3A.

DESCRIPTION OF THE FIGURES AND LEGENDS

[0023] FIG. 1. Relative potency of tamoxifen and its primary and secondary metabolites in the inhibition of aromatase. Test compounds (10 uM) were incubated with 7.5 oM recombinant human aromatasc at 37°C for 30 min. Letrozole (0.1 uM) and vehicle (acetonitrile) were used as positive and negative controls respectively. Data are plotted as means of triplicate incubation with standard deviations. The dotted line represents 100 percent activity that was observed with the vehicle control.

[0024] FIG. 2. Inhibition of aromatase by tamoxifen and its metabolites. Curves represent percent aromatase activity remaining in the presence of a range of concentrations of endoxifen (o), tamoxifen (T), NDMT (·) and 4HT (Δ). Individual points represent the mean of four independent incubations.

[0025] FIG. 3A. Non-competitive inhibition of MFC metabolism by endoxifen. Dixon plot of inhibition of aromatase by endoxifen with MFC concentrations set at 25 (o), 20 (·), 15 (□) and 10O) μΜ.

[0026] FIG. 3B. Eadie-Hofstee plot of inhibition of aromatase by endoxifen with increasing inhibitor concentrations: 0 (A), 1.56 (o), 3.13 (·), 6.25 (□) and 12.5 (♦) μΜ. A range of MFC concentrations was incubated with 7.5 nM recombinant human aromatase for 30 min in the absence and presence of 1.56, 3.13, 6.25 or 12.5 μΜ endoxifen. The rates of HFC generation were determined by measuring fluorescence response as described in the methods section. Individual points represent the mean of duplicate incubations

[0027] FIG. 4 A. Non-competitive inhibition of MFC metabolism by NDMT. Dixon plot of inhibition of aromatase by NDMT with MFC concentrations set at 25 (o), 20 (·), 15 (□) and 10O) μΜ.

[0028] FIG. 4B. Eadie-Hofstee plot of inhibition of aromatase by NDMT with increasing inhibitor concentrations: 0 (A), 3.13 (o), 6.25 (·), 12.5 (□) and 25 (♦) μΜ. A range of MFC concentrations was incubated with 7.5 nM recombinant human aromatase for 30 min in the absence and presence of 3.13, 6.25, 12.5 or 25 μΜ NDMT. The rates of HFC generation were determined by measuring fluorescence response as described in the methods section. Individual points represent the mean of duplicate incubations

[0029] FIG. 5A. Non-competitive inhibition of testosterone metabolism by endoxifen. Dixon plot of inhibition of aromatase by endoxifen with testosterone concentrations set at 8 (o), 4 (·), 2 (□) and 1( ) μΜ.

[0030] FIG. 5B. Eadie-Hofstee plot of inhibition of aromatase by endoxifen with increasing inhibitor concentrations: 0 (Δ), 50 (A), 100 (o), 200 (·), 400 (□) and 600 (♦) μΜ. A range of testosterone concentrations was incubated with 50 nM recombinant human aromatase for 10 min in the absence and presence of 0, 50, 100, 200, 400 or 600 μΜ endoxifen. The rates of estradiol generation were determined by HPLC with UV detection as described in the methods section. Individual points represent the mean of duplicate incubations.

[0031] FIG. 6 A. Competitive inhibition of aromatase by norendoxifen. A range of substrate (MFC) concentrations was incubated with 7.5 nM recombinant human aromatase for 30 min in the absence and presence of norendoxifen. Dixon plot of inhibition of aromatase by norendoxifen with substrate (MFC) concentrations set at 10 (o), 20 (·), 30 (□), 50 (♦) and 100 (Δ) μΜ.

[0032] FIG. 6B. Lineweaver-Burke plot of inhibition of aromatase with increasing norendoxifen concentrations: 0 (□), 10 (·), 25 (o), 50 (♦) and 100 (Δ) nM. Individual points represent the mean of duplicate incubations

[0033] FIG. 7. Selective inhibition of CYP450 isoforms by norendoxifen. In the presence of a range of norendoxifen concentrations, the remaining activity of human placental aromatase (·), human liver CYP3A (o), human liver CYP2C9 (■) and human liver CYP2C19 (A) were determined by measuring the formation rates of metabolites from specific probe drugs and were expressed as percentage of control. Individual points represent the mean of three to four independent incubations

[0034] FIG. 8. Structure-function relationships: stepwise hydroxylation and demethylation of tamoxifen progressively increase the potency of aromatase inhibition. The horizontal open arrows represent the addition of a hydroxyl group. The vertical dark arrows represent the removal of a methyl group. Available compounds tested were: (1) tamoxifen; (2) 4-hydroxy- tamoxifen; (3) 4 '-hydroxy-tamoxifen; (4) 4,4'-dihydroxy-tamoxifen; (5) N-desmethyl- tamoxifen; (6) N-desmethyl-4-hydroxy-tamoxifen or endoxifen, (7) N-desmethyl-4'-hydroxy- tamoxifen; (8) N,N-didesmethyl-4-hydroxy-tamoxifen or norendoxifen.

[0035] FIG. 9. Hypothetical binding mode of E-norendoxifen in the human aromatase active site (PDB ID 3eqm). The ligands are gray, with oxygen depicted in red and nitrogen in blue. The protein is colored green, and the heme is colored magenta. Yellow dashed lines represent the distances between hydrogen bond donors and acceptors. The stereo view is programmed for walleyed (relaxed) viewing

[0036] FIG. 10. Hypothetical binding mode of Z-norendoxifen in the human aromatase active site (PDB ID 3eqm). The ligands are gray, with oxygen depicted in red and nitrogen in blue. The protein is colored green, and the heme is colored magenta. Yellow dashed lines represent the distances between hydrogen bond donors and acceptors. The stereoview is programmed for wall-eyed (relaxed) viewing

[0037] FIG. 11. Hypothetical binding mode of 4,4'-dihydroxytamoxifen in the human aromatase active site (PDB ID 3eqm). The ligands are gray, with oxygen depicted in red and nitrogen in blue. The protein is colored green, and the heme is colored magenta. Yellow dashed lines represent the distances between hydrogen bond donors and acceptors. The stereoview is programmed for wall-eyed (relaxed) viewing.

DESCRIPTION

[0038] For the purposes of promoting an understanding of the principles of the novel technology, reference will now be made to the preferred embodiments thereof, and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the novel technology is thereby intended, such alterations, modifications, and further applications of the principles of the novel technology being contemplated as would normally occur to one skilled in the art to which the novel technology relates are within the scope of this disclosure and what it claim.

Definitions and Abbreviations

[0039] Unless explicitly stated or implicitly intended otherwise the following definitions are used herein, as are the abbreviations listed in Table 1.

[0040] As used herein, unless explicitly stated otherwise or clearly implied otherwise the term 'about' refers to a range of values plus or minus 10 percent, e.g. about 1.0 encompasses values from 0.9 to 1.1

[0041] As used herein, unless explicitly stated otherwise or clearly implied otherwise the terms 'therapeutically effective dose,' 'therapeutically effective amounts,' and the like, refers to a portion of a compound that has a net positive effect on the health and well being of a human or other animal. Therapeutic effects may include an improvement in longevity, quality of life and the like these effects also may also include a reduced susceptibility to developing disease or deteriorating health or well being. The effects may be immediate realized after a single dose and/or treatment or they may be cumulative realized after a series of doses and/or treatments.

[0042] Pharmaceutically acceptable salts include salts of compounds of the invention that are safe and effective for use in mammals and that possess a desired therapeutic activity. Pharmaceutically acceptable salts include salts of acidic or basic groups present in compounds of the invention. Pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzensulfonate, p-toluenesulfonate and pamoate (i.e., l,l'-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Certain compounds of the invention may form pharmaceutically acceptable salts with various amino acids. Suitable base salts include, but are not limited to, aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, and diethanolamine salts. For addition information on some pharmaceutically acceptable salts that can be used to practice the invention pleast reviews such as Berge, et al., 66 J. PHARM. SCI. 1-19 (1977), Haynes, et al, J. Pharma. Sci., Vol. 94, No. 10, Oct. 2005, pgs. 2111-2120 and the like.

Table 1. Some abbreviations used herein.

[0043] The biochemical mechanism of action of tamoxifen in the treatment of breast cancer is widely understood to involve two active metabolites, 4-hydroxy-N-desmethyl-tamoxifen (endoxifen) and Z-4-hydroxy-tamoxifen (4HT). These metabolites are approximately 100 times more potent, relative to the parent drug, as antagonists of estrogen binding to the estrogen receptor and as inhibitors of estrogen- stimulated growth in sensitive breast cancer cell lines. What is thought to be the principal active metabolite, endoxifen is produced via metabolism from N-desmethyl-tamoxifen (NDMT), by a genetically polymorphic enzyme, CYP2D6. This has led many clinical investigators to test the possibility that the CYP2D6 genotype might be a useful biomarker of tamoxifen efficacy. The results of these efforts have produced mixed results. While some investigators have shown a association between the CYP2D6 poor metabolizer genotype and increased recurrence of breast cancer in some settings, other investigators, using different trial designs and alternative study approaches have either failed to show any association, or have shown an association in the opposite direction. As a result, the clinical utility of a CYP2D6 genotype guided approach to predicting response to tamoxifen has not been clearly demonstrated. The closely related concept that endoxifen or 4HT concentrations in the blood might predict the outcome of tamoxifen therapy in individual patients has also not been validated in any clinical study.

[0044] The side effects of tamoxifen are also hard to predict. This is important because many women experience hot flashes, muscle aches and other symptoms that limit their compliance with treatment, and clearly result in increased rates of breast cancer recurrence.

[0045] No relationship between the concentrations of the estrogen receptor modulating metabolites of tamoxifen and the incidence or severity of side effects has been reported. Furthermore, although CYP2D6 genotype is clearly associated with the concentrations of these active metabolites, it has not been consistently shown to predict hot flashes experienced by patients taking tamoxifen.

[0046] One possible explanation for the lack of association between active metabolite concentrations and the clinical effects of tamoxifen may be the involvement of mechanisms other than estrogen receptor antagonism. Among these, aromatase inhibition would appear potentially important since drugs that block aromatase activity have been shown to be effective treatments for breast cancer. [0047] In the context of breast cancer, currently marketed aromatase inhibitors are generally used to treat post-menopausal women who have tumors that are "estrogen receptor positive" by widely used clinical assays. Nearly 80% of all newly diagnosed breast cancers are positive for the estrogen receptor (ER). Adjuvant anti-estrogen, or "endocrine" therapy, with the selective estrogen receptor modulator, tamoxifen, reduces recurrence and mortality in women with ER positive breast cancer by 40%. Over the last decade, large definitive trials have demonstrated that therapy with aromatase inhibitors (AIs) is more effective than tamoxifen treatment in both the metastatic and adjuvant settings, but the absolute benefit relative to tamoxifen is only 2-4%.

[0048] There are three commercially available aromatase inhibitors: the azoles, anastrozole (Arimidex™) and letrozole (Femara™), which are potent competitive inhibitors, and the steroidal compound, exemestane (Aromasin™), which is a mechanism-based inhibitor. All three of these compounds result in decreased circulating estrogen concentrations by at least 10-fold compared to concentrations before treatment in untreated postmenopausal women. Despite their efficacy, it is clear that many patients with ER positive breast cancer do not benefit from an adjuvant aromatase inhibitor, even if their tumor is destined to recur, and more than half of women with ER positive breast cancer still develop incurable metastastic breast cancer. It follows that there remains a need for better therapies or improved administration of existing therapies.

[0049] Therapy with the currently marketed aromatase inhibitors results in marked side effects that make the drugs difficult to tolerate for the 2- 5 year required for effective breast cancer treatment. Toxicities are one of the major reasons for discontinuation of AIs, and the most common adherence-limiting toxicity is the AI-associated musculoskeletal syndrome, or AIMSS.

[0050] AIMSS syndrome consists of a constellation of musculoskeletal symptoms, including generalized arthralgias, trigger finger, digital stiffness, carpal tunnel syndrome, or tendinitis/tendinopathy that occur in the absence of any alternative reason for development of these symptoms, such as trauma, pre-existing rheumatoid arthritis, or other definable causes. Earlier studies suggest that AIMSS is the reason for discontinuation in 10-20% of all patients taking an aromatase inhibitor and in the majority of those who discontinue therapy. In our recently completed trial comparing exemestane vs. letrozole, AIMSS was the reason for discontinuation in 70%> of those who stopped the drug. These data are consistent with the largest patient survey published, which showed that -75% of patients who discontinued aromatase inhibitor therapy did so because of AIMSS.

[0051] Accordingly, there is a significant need for more tolerable aromatase inhibitors that are able to treat breast cancer and other conditions, but that have fewer, or better still, no treatment-limiting side effects. The compounds reported on herein are well recognized as metabolites of tamoxifen but have not been previously identified as aromatase inhibitors. Tamoxifen itself is used as a selective estrogen receptor modulator in the treatment of breast cancer. Tamoxifen itself has effects and side-effects that are similar to some of those of the aromatase inhibitors. This commonality of effect suggests a common mechanism of action, and therefore also suggests that tamoxifen metabolites may be sufficiently potent as aromatase inhibitors to have clinical effects at concentrations routinely experienced by patients taking tamoxifen.

[0052] While the commonality of these side effects may reflect a shared biochemical mechanism, the possibility that treatment with tamoxifen inhibits aromatase activity has not been systematically investigated. Of note, tamoxifen has been shown to significantly lower estrogen in post-menopausal women.

[0053] One possibility is that the clinical effects of tamoxifen may be mediated in part by actions of tamoxifen or its metabolites as aromatase inhibitors. To investigate this possibility, the ability of tamoxifen and its primary human metabolites to inhibit the activity of aromatase in vitro were tested. The data disclosed herein suggests that some tamoxifen metabolites are able to inhibit aromatase with potencies in the nanomolar range, entirely consistent with the potency of the known aromatase inhibitors. The compounds describe herein have the potential to provide alternative treatment options, or to be more effective medications than the current aromatase inhibitors especially if they are more tolerable.

[0054] Suitable aromatase inhibitors for use in the present invention are a series of compounds that have triphenylalkene structure with side chain(s) on the phenyl group(s) that are oxyalkanes or oxyalkenes and that terminate in an unsubstituted or mono-substituted amine, including a geometric isomer, a stereoisomer, a pharmaceutically acceptable salt, an ester thereof or a metabolite thereof. Such structures include, but are not limited to the N-demethylated metabolites of tamoxifen, such as N-desmethyltamoxifen and N,N-didesmethyl-4- hydroxytamoxifen. Among these compounds, 4-hydroxylated derivatives such as N-desmethyl- 4-hydroxytamoxifen (endoxifen) and norendoxifen may be used as drugs with combined effects involving both aromatase inhibition and selective estrogen receptor modulation. This range of structures and their attendant pharmacologic potencies provides a reasonable pharmacophore upon which to build novel aromatase inhibitors that operate via this new biochemical mechanism.

[0055] The ability of a series of compounds of triphenylalkene structure to inhibit the activity of the human aromatase enzyme (recombinant CYP19) in vitro was tested. Some of the compounds tested included tamoxifen, (Z)-4-hydroxytamoxifen, N-desmethyltamoxifen, (E/Z)- N-desmethyl-4-hydroxytamoxifen (endoxifen), (E/Z)-N-desmethyl-4'-hydroxytamoxifen, (E/Z)- N,N-didesmethyl-4-hydroxytamoxifen, and tamoxifen-N-oxide. The activity of aromatase was determined by measuring the conversion rate of a fluorometric substrate, 7-methoxy-4- trifluoromethylcoumarin (MFC), to its fluorescent metabolite, 7- hydroxytrifluoromethylcoumarin (HFC), in the presence and absence of multiple concentrations of tested inhibitors as described in methods section below. The biochemical mechanisms of inhibition by these compounds were documented and their inhibitory potency was compared.

[0056] When the inhibitor concentration was set at 10 μΜ, N-desmethyltamoxifen, endoxifen, (E/Z)-N-desmethyl-4'-hydroxytamoxifen, (E/Z)-N,N-didesmethyl-4- hydroxytamoxifen, and tamoxifen-N-oxide were able to inhibit aromatase with a range of potencies (FIG. 1). Letrozole, a known potent aromatase inhibitor, was used as positive control. No appreciable inhibition by tamoxifen and (Z)-4-hydroxytamoxifen was observed at this concentration.

[0057] Among these inhibitors, N,N-didesmethyl-4-hydroxytamoxifen, endoxifen and N- desmethyltamoxifen were further characterized for their mechanisms of action on aromatase. A range of MFC concentrations were incubated with 7.5 nM recombinant human aromatase for 30 min in the absence and presence of 10, 25, 50 or 100 nM N,N-didesmethyl-4-hydroxytamoxifen. Details of incubation conditions and quantitative assays are provided in the methods section. Individual points represent the mean of duplicate incubations.

[0058] A range of MFC concentrations were incubated with 7.5 nM recombinant human aromatase for 30 min in the absence and presence of 1.56, 3.13, 6.25 or 12.5 μΜ endoxifen. Details of incubation conditions and quantitative assays are provided in the methods section. [0059] A range of MFC concentrations were incubated with 7.5 nM recombinant human aromatase for 30 min in the absence and presence of 3.13, 6.25, 12.5 or 25 μΜ N- desmethyltamoxifen. Details of incubation conditions and quantitative assays are provided in the methods section.

[0060] Detailed kinetic analysis of the inhibition by these inhibitors was carried out as described in the methods section below. When a range of inhibitor concentrations was tested, N,N-didesmethyl-4-hydroxytamoxifen and letrozole were found to be potent inhibitors of aromatase with IC₅₀ values of 30 nM and 5 nM, respectively. Under the same conditions, endoxifen and N-desmethyltamoxifen were found to be relatively weaker inhibitors with IC₅₀ values of 6.1 μΜ and 20.7 μΜ, respectively (Table 2). When inhibitor concentration was increased to 100 μΜ, 4-hydroxytamoxifen and tamoxifen also showed inhibitory effects on aromatase activity (data not shown). The model estimated IC₅₀ values for 4 -hydroxytamoxifen and tamoxifen were 531 μΜ and 986 μΜ, respectively (Table 2).

[0061] When aromatase inhibition by these inhibitors was tested using a range of fluorometric substrate concentrations, K; values for N,N-didesmethyl-4-hydroxytamoxifen, endoxifen and N-desmethyltamoxifen were found to be 35 nM, 4 μΜ and 15.9 μΜ, respectively (Table 3). Further kinetic analysis using Eadie-Hofstee plots and/or Lineweaver-Burk plots indicated that N,N-didesmethyl-4-hydroxytamoxifen acts as a competitive inhibitor of aromatase, whereas endoxifen and N-desmethyltamoxifen appeared to be non-competitive inhibitors (data not shown).

Table 2. IC₅₀ values of inhibitors of recombinant human aromatase.

Table 3. K; values of inhibitors of recombinant human aromatase.

[0062] Referring now to FIGs. 1 and 8. The basic triphenylalkene structure of tamoxifen is shown as an example, as it undergoes progressive demethylation or hydroxylation. The horizontal open arrows represent the addition of a hydroxyl group, while the vertical, dark arrows represent the removal of a methyl group. As methyl groups are progressively removed, inhibitory potency appears to increase, while the addition of a single hydroxyl group also increases potency to some extent.

[0063] Surprisingly, a comparison of the chemical structures of various novel aromatase inhibitors to the structures of known high-affinity aromatase substrates and inhibitors and an examination of the structural relationships between these compounds (FIG. 8), suggests a novel family of effective aromatase inhibitors.

[0064] A generic formula for a family of effective aromatase inhibition may be brought about by compounds based upon a triphenylalkene structure with side chain(s) on the phenyl group(s) that are oxyalkanes or oxyalkenes, and that terminate in an unsubstituted or mono- substituted amine. This basic structure can be modified to provide a series of potential aromatase inhibitors for use in various conditions, and could provide a wider range of therapeutic choices than those that are currently available.

[0065] Inhibition of aromatase by tamoxifen, NDMT and the two metabolites known to be active at the estrogen receptor: 4HT and endoxifen was tested by following the fluorescence generated during incubation with aromatase and a fluorometric substrate, MFC. FIG. 1 shows that endoxifen and NDMT inhibited aromatase with higher potency than tamoxifen or 4HT. When substrate concentration was set at 25 μΜ, endoxifen and NDMT exhibited IC50S of 6.1 μΜ and 20.7 μΜ, respectively, while tamoxifen and 4HT were estimated to have IC50S of 986 μΜ and 531 μΜ, respectively (Table 4). Under the same conditions, letrozole was used as a positive control and had an IC50 of 5.4 nM. When the endogenous substrate of aromatase, testosterone, was included as a competing substrate, an IC50 of 0.33 μΜ was observed.

Table 4 IC50 values of inhibitors of recombinant human aromatase

Inhibitor Endoxifen NDMT 4HT Tamoxifen Letrozole Testosterone

IC₅₀ (μΜ) 6.1 20.7 531 986 0.0053 0.33

IC50 values were determined when MFC and aromatase concentrations were set at 25 μΜ and 7.5 nM respectively, as described in the methods section. Table 5 Effect of endoxifen and NDMT on Ks_app and V_ma5a- of recombinant human aromatase

Inhibitor (μΜ) K_Sapp (μΜ) V_maxi (pmol/min/pmol P450)

Endoxifen

0 58.5 4.05

1.56 60.8 3.56

3.13 57.8 2.72

6.25 61.9 2.03

12.5 66.2 1.22

NDMT

0 52.4 4.43

3.13 54.2 4.09

6.25 51.9 3.23

12.5 52.4 2.53

25 61.8 2.03

Ks_app, the apparent Michaelis constant. V_maxz-, the apparent maximum reaction rate. Ks_app and m_a5u values were determined using Lineweaver-Burke plot as described in the methods section.

[0066] To explore further the mechanism of the inhibition by endoxifen and NDMT, the inhibition by these metabolites was tested across a range of fluorometric substrate

concentrations. The data were plotted as Dixon and Eadie-Hofstee plots (FIGs. 2 to 5). The profile of the lines on the resulting Dixon plots: straight lines intersecting at a common point on the x-axis, is consistent with non-competitive inhibition by endoxifen and NDMT (FIGs. 3A and 4A). The parallel relationship of the lines in the Eadie-Hofstee plots is consistent with decreasing maximum enzyme activity V_maxz and unchanged substrate equilibrium dissociation constant Ks_app as inhibitor concentration was increased (FIGs. 3B and 4B), observations that were also consistent with a non-competitive mechanism. The data in these assays indicate a K; for endoxifen of 4.0 μΜ, and a K_; for NDMT of 15.9 μΜ.

[0067] To study the inhibitory mechanism using an alternative approach, the same data were plotted using Lineweaver-Burke plots. The data obtained also indicate that both endoxifen and NDMT decreased the apparent V_max (V_maxz), while leaving the apparent K_m (Ks_app) unchanged (Table 4), once more consistent with non-competitive inhibition.

[0068] The ability of the metabolites that inhibited MFC metabolism by aromatase to inhibit testosterone metabolism to estrogen was tested. In order to do so, higher concentration of aromatase (50 nM) was incubated with multiple testosterone concentrations chosen to be within the linear range around the K_m (4 μΜ) in order that the generation of estrogen could be confidently detected. When a range of concentrations of endoxifen were incubated with testosterone and aromatase under these conditions, endoxifen inhibited the generation of estrogen with a Ki of 178 μΜ (FIG. 5A), with kinetics examined using Dixon and Eadie-Hofstee plots that again indicate non-competitive inhibition (FIGs. 5A and 5B). Equivalent experiments with NDMT indicated that NDMT was a weaker inhibitor than endoxifen (data not shown), but experiments for Ki determination could not be carried out due to its relative insolubility at higher concentrations which were desired.

[0069] In order to test whether these observations applied in a more physiologically relevant system, the ability of tamoxifen, 4HT, endoxifen and letrozole to inhibit the conversion of MFC to HFC by human placental aromatase was also tested. Under the same conditions as used in MFC incubations with recombinant enzyme, and with the substrate concentration set at 25 μΜ, the selective inhibitor letrozole (20 nM) completely inhibited aromatase activity. Endoxifen was able to inhibit placental aromatase with an IC₅₀ of 5 μΜ. Consistent with our previous data, NDMT inhibited placental aromatase with a weaker potency while tamoxifen and 4HT did not inhibit at concentrations up to 50 μΜ (data not shown).

[0070] Since the K for endoxifen determined at this higher enzyme concentration (50 nM) using testosterone as substrate, was notably higher than that obtained at 7.5 nM of aromatase using MFC as substrate, whether the experimental conditions used in the testosterone

incubations, including in particular the increased enzyme concentration would result in an increase in the observed IC₅₀ of endoxifen was tested. Under the same conditions as those when an enzyme concentration of 50 nM was used, an IC₅₀ of 95 μΜ for endoxifen was observed in inhibition of MFC metabolism (data not shown), which was 15 to 16 fold higher in comparison to 6.1 nM as shown in Table 4.

[0071] When tamoxifen, 4HT, NDMT or endoxifen was pre -incubated with aromatase for 5, 10, 15 and 20 min to test the possibility of irreversible inhibition, no decrease in MFC metabolism to HFC or testosterone metabolism to estradiol relative to control was observed (data not shown). These data suggest that the inhibition of aromatase observed with endoxifen and NDMT is a reversible process.

[0072] Documented herein is the inhibition of human aromatase by two tamoxifen metabolites: endoxifen and NDMT. The inhibitory concentrations are high relative to those seen in human serum, but not to those reported in tissue. Under conditions of low enzyme

concentration (7.5 nM) in vitro, the Ki of the most potent metabolite studied, endoxifen was 4.0 μΜ. While the serum concentrations of endoxifen in humans are in the 10 - 150 nM range.

Tissue concentrations of endoxifen are higher, especially in breast tumors, where they appear to be 10 to 100 times more, i.e. above 10 μΜ. Furthermore, in rats the ratio of endoxifen concentrations in uterus to those in serum has been reported to be at least 20: 1 , and to be at least 500: 1 between lung and serum. These high tissue concentrations are consistent with the large apparent distribution volume for tamoxifen, the parent drug, which is about 50 to 60 liters/kg in humans, indicating that most of the drug (99.9%) is present in peripheral compartments, and suggesting extensive tissue binding. This wide tissue distribution, and the relatively long apparent half lives of NDMT and endoxifen (~14 and 44 days respectively) in humans treated with tamoxifen are consistent with an extended period of tissue exposure. Sustained aromatase inhibition in vivo by these metabolites would therefore seem a possibility.

Since the initial observations were carried out using an artificial, fluorometric substrate for aromatase, the physiologic relevance of these data was confirmed by showing that endoxifen was also able to inhibit metabolism of the endogenous substrate of aromatase, testosterone. The inhibition of testosterone metabolism to estradiol was studied under conditions involving higher aromatase concentrations so that the generation of estradiol could be quantitatively monitored. A Ki of 178 μΜ was observed for endoxifen under incubation conditions that included a 6.7 fold higher enzyme concentration (50 nM). Of note, two observations suggest that in conditions involving lower aromatase concentrations, testosterone metabolism could be inhibited by lower concentrations of endoxifen. First, in the two experimental conditions tested, the IC₅₀ of endoxifen for the same substrate, MFC, differed by 15 - 16 fold (6.1 μΜ at 7.5 nM aromatase, compared to 95 μΜ at 50 nM aromatase), suggesting a lower at lower enzyme concentrations. Second, we observed that the K_m for testosterone in this system was 4 μΜ, significantly higher than that (0.2 μΜ) reported in placental microsomes. It follows that under physiologic conditions, where the concentrations of aromatase and of testosterone are lower than those used in vitro, inhibition is likely to occur at relatively low inhibitor concentrations.

[0073] While tamoxifen itself was not able to inhibit aromatase, many tamoxifen metabolites were capable of doing so. FIG. 1 shows the relative potency of tamoxifen and its available metabolites as AIs. Among the primary metabolites, N-desmethyl-tamoxifen, ^-hydroxy- tamoxifen and tamoxifen-N-oxide were all relatively weak inhibitors. Among the secondary metabolites of tamoxifen, endoxifen and 4, 4'-dihydroxy-tamoxifen were more potent inhibitors than the primary metabolites, while norendoxifen was the most potent of all the inhibitors tested, and the only metabolite that completely inhibited enzyme activity at an inhibitor concentration of 10 μΜ. The inhibitory potency order of the tested compounds was as follows: norendoxifen » 4,4'-dihydroxy-tamoxifen > endoxifen > N-desmethyl-tamoxifen, N-desmethyl-4'-hydroxy- tamoxifen, tamoxifen-N-oxide, 4'-hydroxy-tamoxifen, N-desmethyl-droloxifene > 4-hydroxy- tamoxifen, tamoxifen. Consideration of the structure-activity relationships generated by these data makes clear that a number of consistent relationships exist. As methyl groups are progressively removed, inhibitory potency increased substantially, while the addition of a single hydroxyl group also increased potency (FIG. 1 and 8).

[0074] To explore further the mechanism of the inhibition by the most potent AI identified, norendoxifen, we tested inhibition across a range of fluorometric substrate concentrations. The data were plotted as Dixon and Lineweaver-Burke plots. The lines on the resulting Dixon plot intersecting at a common point indicated a K_z of 35 nM (FIG. 6A). The profile of the lines on the resulting Lineweaver-Burke plot: straight lines intersecting at a common point on the y-axis was consistent with a competitive mechanism of inhibition by norendoxifen (FIG. 6B). In order to explore the stereoselectivity of norendoxifen, the potency of the purified E-enantiomer of norendoxifen was tested. Under the same experimental conditions, the IC₅₀ values for E- norendoxifen and the unseparated mixture were both approximately 30 nM (data not shown).

[0075] In order to test whether the inhibitory effect of norendoxifen on aromatase extends to other CYP enzymes, the ability of this compound to inhibit other important drug-metabolizing CYP enzymes was tested, including CYP2B6, CYP2C9, CYP2C19, CYP2D6 and CYP3A. When CYP2B6 and CYP2D6 were tested, no substantial inhibition by norendoxifen was observed at concentrations up to 1 μΜ. Three experimental systems were used to test inhibition of aromatase (CYP 19), CYP2C9, CYP2C19 and CYP3A by norendoxifen: drug incubations with recombinant CYP isoforms, pooled placental microsomes or pooled human liver microsomes. Initially, when recombinant CYP isoforms were used, norendoxifen inhibited CYP 19, CYP2C9 and CYP2C19 with IC₅₀ values of 30, 95 and 61 nM respectively. These data did not suggest obvious CYP isoform selectivity. Since the enzyme concentrations and configurations present in recombinant systems may not represent the dynamic multi-enzyme system present in vivo, CYP3 A was not tested in this system. Instead, the selectivity of norendoxifen was further characterized using pooled placental and pooled human liver microsomes under more

physiologic conditions and with similar total protein concentrations. Norendoxifen inhibited placental aromatase with an IC₅₀ value of 90 nM, while it inhibited human liver CYP2C9 and CYP3A with IC₅₀ values of 990 and 908 nM respectively (FIG. 7). Inhibition of human liver CYP2C19 by norendoxifen appeared even weaker, with less than 25% inhibition observed at concentrations up to 5 μΜ (FIG. 7).

[0076] A molecular modeling study was performed in order to rationalize the aromatase inhibitory activities of tamoxifen metabolites. The compounds were docked into the active site of aromatase (PDB ID 3eqm) using GOLD software and the energies of the complexes were minimized using the Amber force field and Amber charges. The binding free energies were calculated and are summarized together with the experimental aromatase IC₅₀ values in Table 1. These estimated free energies appeared positively associated with the relative inhibitory potencies observed. The docking and energy minimization procedure was validated by reproducing the published crystal structure of aromatase-androstenedione complex by extracting the ligand structure and then docking it back into the aromatase active site, merging the highest- scored binding pose with the protein, and then minimizing the complex energy following the same protocol used with other tamoxifen metabolites. The root mean standard deviation between the structure of the newly generated complex derived from molecular modeling and the original crystal structure (PDB ID 3eqm) was 1.73 A.

[0077] In order to characterize the activity of the most potent inhibitor, one that might represent a lead compound to guide future rational drug design, the hypothetical binding modes of the norendoxifen isomers is presented in FIGs. 9 and 10. Both isomers have similar binding modes. According to the models, the phenolic hydroxyl groups of both isomers bind to the carbonyl group of Met374. The other oxygen atoms of both forms are calculated to exist near the iron atom; however, the ether oxygen atom of the E isomer was calculated to be 0.8 A closer to the iron atom with better directionality for binding than the Z form. A hydrogen bond is apparent between the terminal aliphatic amino group in the E form and the carbonyl group of Ala306. A similar interaction is not predicted for the Z form, in which the terminal aliphatic amino nitrogen atom was calculated to at least 5 A away from the closest atom that it could hydrogen bond to.

[0078] On the other hand, the unsubstituted phenyl ring and the ethyl moiety in both E and Z forms are surrounded by hydrophobic residues including Phe221, Leu477, Val370, Ile70, and the benzene ring of Trp224. In addition, the phenyl ring that contains the hydroxyl group is calculated to from a possible side-to-face stacking interaction with Phel34 in both isomeric forms. A comparison of the two complexes reveals that the ethyl and phenyl groups switch locations, but the two remaining phenyl rings that contain hydrogen bonding substituents maintain their positions.

[0079] Table 6 Calculated binding free energy for most stable docking poses (MM-BPSA) and the experimental IC₅₀ values of compounds

[0080]

Calculated Binding Free Energy . _^ , _T ^

Compound ^{& &J} Experimental IC₅₀

(Kcal/mol)

Z-norendoxifen -65.2

30 nM*

E- norendoxifen -58.9

Z-endoxifen -61.0

6 μΜ*

E-endoxifen -60.1

N-desmethyl-tamoxifen -59.6 20 μΜ

Z-4-hydroxy-tamoxifen -50.5 530 μΜ

Tamoxifen -50.6 985 μΜ

values were determined when mixtures of unseparated E and Z isomers were tested.

[0081] Aromatase inhibition was observed to occur via a non-competitive mechanism, which is consistent with an allosteric interaction with aromatase. This may explain why it was possible for endoxifen to effectively inhibit testosterone metabolism, although the observed IC₅₀ value for inhibition of MFC metabolism by testosterone was 19-fold lower than that of endoxifen (Table 4). The structure of the active catalytic site of aromatase and its interactions with androgens has been well studied. However, potential interactions at other drug binding sites have not been considered until now. It is possible that the allosteric inhibition occurs via a site remote from the catalytic site, or that it occurs via interaction of two drugs that bind differently within the active site. Of note, mutation of a site distant from the substrate binding site has been shown to increase enzyme activity and reduce the susceptibility to inhibition of aromatase by aminoglutethimide. These observations raise the possibility that an allosteric mechanism might contribute to the pharmacologic regulation of aromatase and could be exploited to modulate aromatase activity for therapeutic benefit.

[0082] The interpretation of this study is limited by the difficulty of inferring drug concentrations at the effect site in vivo, given the acknowledged gradient between serum and tissue concentrations. As a result, the potency of this mechanism in vivo is unclear. In addition, although NDMT is a less potent inhibitor, it exists in ~ 10-fold higher serum concentrations than endoxifen in humans. Therefore, the relative contributions of endoxifen and NDMT to aromatase inhibition in vivo remain unclear.

[0083] These observations could help explain a number of currently unexplained observations. First, these data are consistent with the observation that estrogen concentrations decreased on average in post-menopausal women being treated with tamoxifen. Second, it is possible that inhibition of aromatase by tamoxifen might help explain why tamoxifen causes musculoskeletal pain, similar to that commonly experienced by patients taking aromatase inhibitors. This side effect of tamoxifen appears debilitating and prominent in Asian women, who experience few hot flashes during tamoxifen treatment. It is possible that, in the sub- population of post-menopausal women in whom musculoskeletal pain is a severe side effect of tamoxifen, aromatase inhibition by its metabolites is more prominent. Third, the data may help explain the inconsistency in observed associations between CYP2D6 genotype and outcomes in patients with breast cancer. If aromatase inhibition contributes to the action of tamoxifen, then it is possible that this inhibition may confound simple associations between endoxifen

concentrations and clinical outcomes. In addition, mechanistic studies which employ only 4HT, an estrogen receptor modulator that is not an aromatase inhibitor may inadequately represent tamoxifen action in vivo.

[0084] Tamoxifen metabolites, including endoxifen and N-desmethyl-tamoxifen can act as AIs in vitro with K_z values of 4 and 15.9 μΜ respectively. As disclosed herein, norendoxifen is a potent and selective inhibitor of human aromatase with a IQ value in the nanomolar range, close to the potency of the positive control used: letrozole (IC₅₀ of 5.3 nM), which is the most potent AI that is available for clinical use. Norendoxifen also appears to be a selective AI. When tested for the inhibition of important drug metabolizing CYP enzymes, it did not inhibit CYP2B6 or CYP2D6 at all, and was at least 10-fold less potent as an inhibitor of CYP2C9, CYP2C19 and CYP3 A. The ability of norendoxifen to inhibit aromatase in vivo is at present unclear and deserves further study.

[0085] Although norendoxifen is a known metabolite of tamoxifen in humans, little is known about its tissue concentrations or its contribution to tamoxifen effects. It is a minor metabolite of tamoxifen that exists at notably lower concentrations than the parent drug or its major metabolites, but these data make clear that it is a much more potent inhibitor of aromatase than the other known inhibitory tamoxifen metabolites, endoxifen and N-desmethyl-tamoxifen. In as much as these two metabolites may contribute to tamoxifen action via aromatase inhibition, it is equally possible that norendoxifen contributes significantly to the clinical effects of tamoxifen. In addition, endoxifen itself is being developed as a drug. Accordingly, the role of norendoxifen, the demethylated metabolite, in endoxifen action may be even more important.

[0086] The metabolism of tamoxifen is complex and so its ultimate effects reflect the aggregation of the actions of multiple metabolites on the estrogen receptors, on aromatase and also possibly via other mechanisms that have been reported. Data from the definitive trail that compared anastrozole, tamoxifen, and the combination of both drugs in the adjuvant treatment of breast cancer indicated the effects of tamoxifen combined with an AI are inferior to those of an AI alone. While this suggests that the aggregated aromatase inhibition by tamoxifen metabolites is not additive with that of anastrozole, in fact anastrozole is extremely potent and it may be very difficult to increase its overall benefit by further inhibition. That said, this does not obviate the possibility that aromatase inhibition by norendoxifen or other metabolites contributes to tamoxifen effects when it is used alone. The inferiority observed could be due to worse compliance with anastrozole and tamoxifen in the combination arm relative to the anastrozole arm, or to separate and deleterious effects of tamoxifen or its metabolites.

[0087] As reported herein, a number of tamoxifen metabolites have activity as AIs with a wide range of potencies. Although these studies are limited to the commercially available tamoxifen metabolites, they are sufficient to allow the exploration of relationships between the structures of the compounds we tested and their function. The data indicate that stepwise hydroxylation and demethylation of tamoxifen both resulted in progressive increases in inhibitory potency (FIGs. 1 and 8). The activities of 4-hydroxytamoxifen (FIG. 8, Structure 2, IC₅₀ 530 μΜ), endoxifen (Structure 6, IC₅₀ 6 μΜ), and norendoxifen (Structure 8, IC₅₀ 30 nM) show that sequential N-demethylation results in a very significant increase in aromatase inhibitory potency. The molecular models (FIGs. 9-11) document limited space available in the ligand binding site surrounding the amine, and the decrease in activity observed with the presence of more methyl groups can be attributed to steric factors. The models displayed in FIGs 9 and 10, indicate that the amino groups of both isomers are hydrogen bonded to the carbonyl oxygen atom of Ala306. This suggests that the loss of activity seen with the methylation of the amine may also result from a decrease in its capacity to act as a hydrogen bond donor toward the Ala306 carbonyl oxygen. The images displayed in FIGs 9 and 10, also indicate that the unsubstituted phenyl rings and ethyl groups of the double bond isomers of norendoxifen are buried in hydrophobic cavities. In the in vitro experimental data, the E and ZIE mixture of norendoxifen appeared to have the same enzyme inhibitory activities suggesting that the activities of the Z and E isomers are equal. The similar activities of the two isomers and the perspective offered by the molecular models, suggest that the locations of the phenyl and ethyl groups can be switched with virtually no change in activity. The ethyl and unsubstituted phenyl groups therefore appear to contribute to enzyme affinity through the presence of general hydrophobic and dispersion (van der Waals) interactions, as opposed to being due to specific interactions with particular amino acid side chains of the enzyme. Overall, the data suggest that the double bond stereochemistry in this series of AIs may not have a large impact on biological activity, although smaller effects remain possible, since in silico, the molecular models did indicate a greater calculated binding free energy for the Z isomer (Table 6).

[0088] Comparison of the activities of N-desmethyltamoxifen (FIG. 8, Structure 5, IC₅₀ 20 μΜ) and endoxifen (Structure 6, IC₅₀ 6 μΜ) documents a positive contribution made by the 4- hydroxyl group. The molecular models suggest that this may reflect hydrogen bonding of the phenol with the carbonyl of Met374. Furthermore, comparison of the activities of 4- hydroxytamoxifen (Structure 2) and 4,4'-dihydroxytamoxifen (Structure 4) represented in Figure 1 indicates that the 4'-hydroxyl group makes a large positive contribution to the activity. The hypothetical model of the complex of human aromatase with 4,4'-dihydroxytamoxifen (Structure 4) suggests that the 4'-hydroxyl groups contribute to the affinity of the ligand through hydrogen bonding with the carbonyl oxygen of Asp309 (FIG. 11).

[0089] These results illustrate that modifications to the basic triphenylalkene structure of tamoxifen that preserve hydrogen bonding to the Ala306, Met374 and Asp309 residues might be a valuable approach in the development of new AIs, and that norendoxifen is able to serve in this context as a lead compound. Of note, the specific interactions noted here with Ala306 and Met374 were also noted as being key to favourable interactions between aromatase and a series of other ligands.

[0090] In this context it is important to note that norendoxifen is the demethylated metabolite of endoxifen, a widely recognized and potent estrogen receptor modulator. It follows that norendoxifen may also act as an estrogen receptor ligand, that is able to modulate estrogen receptor signalling. Norendoxifen or its derivatives may therefore be valuable as alternative AIs that are able to mitigate the debilitating musculoskeletal toxicities experienced by breast cancer patients via tissue specific mechanisms involving estrogen receptor signalling. This possibility deserves further investigation.

[0091] Overall, the data disclosed herein emphasizes that tamoxifen and related molecules such as norendoxifen and endoxifen and may have multiple pharmacologic effects in the treatment of breast cancer that are mediated by their active metabolites. These data also illustrate the effects of multiple tamoxifen metabolites on aromatase. Most notable among these is norendoxifen, which is a potent and selective inhibitor. The structure-function relationships characterized and the molecular modelling carried out suggest that norendoxifen merits further investigation as a clinical aromatase inhibitor, and may be able to serve as a lead compound for the rational design of novel aromatase inhibitors.

EXPERIMENTAL

Materials and Methods Chemicals and Reagents

[0092] Tamoxifen, N-desmethyltamoxifen, Z-4-hydroxytamoxifen, endoxifen, and letrozole were purchased from Toronto Research Chemicals Inc. (North York, ON, Canada). 17-β- estradiol, testosterone, β-NADP, glucose-6-phosphate dehydrogenase, and glucose-6-phosphate were purchased from Sigma-Aldrich (St. Louis, MO). Magnesium chloride was purchased from Fisher Scientific (Pittsburgh PA). All drug solutions were prepared by dissolving each compound in methanol or acetonitrile, and were stored at -20 °C. Tamoxifen and its metabolites were prepared under dim light and in brown tubes to minimize photodegradation. All HPLC-grade reagents and chemicals used for mobile phase and buffers were obtained as previously described Lu WJ, Bies R, Kamden LK, Desta Z, Flockhart DA (2010) Methadone: a substrate and mechanism-based inhibitor of CYP19 (aromatase). Drug Metab Dispos 38 (8): 1308-1313. doi: 10.1124/dmd.110.032474.

[0093] Pooled human liver microsomes (HLMs) and the cytochrome P450 (CYP) inhibitor screening kits for CYP 19, 2C9, 2C19 and 2D6 were purchased from BD Biosciences (San Jose, CA). Placental microsomal preparations were a generous gift from Dr Mahmoud S. Ahmed at the University of Texas Medical Branch, Galveston.

Microsomal preparations

[0094] Baculo virus-insect cell-expressed human CYP 19 (with oxidoreductase) and the CYP19/MFC high throughput inhibitor screening kit were purchased from BD Biosciences (San Jose, CA). All microsomal preparations were stored at -80°C.

Measurement of Inhibition of Aromatase in vitro

[0095] The activity of aromatase is determined by measuring the conversion rate of a fluorometric substrate, 7-methoxy-4-trifluoromethylcoumarin (MFC), to its fluorescent metabolite, 7-hydroxytrifluoromethylcoumarin (HFC). Experimental procedures are consistent with the methodology described for high-throughput screening of a human cytochrome P450 inhibitor. Stresser DM: High-throughput screening of human cytochrome P450 inhibitors using fluorometric substrates. In Optimization in drug discovery: in vitro methods. Edited by Caldwell ZYGW; 2004: 215-230. All incubations are carried out using incubation times and protein concentrations that are within the linear range for reaction velocity. Experiments involving tamoxifen and its metabolites are carried out under dim light to minimize photodegradation. MFC and inhibitors are prepared in acetonitrile. A series of concentrations of inhibitor in a volume of 4 μΐ are mixed with 96 μΐ of NADPH-Cofactor Mix (16.3 μΜ NADP, 828 μΜ glucose-6-phosphate, 828 μΜ MgCl₂, and 0.4 U/ml glucose 6-phosphate dehydrogenase), and prewarmed for 10 min at 37°C. MFC and recombinant human CYP 19 are mixed with 0.1 M potassium phosphate buffer (pH 7.4), and then added to an Enzyme/Substrate Mix. Reactions are initiated by adding 100 μΐ of Enzyme/Substrate Mix to bring the incubation volume to 200 μΐ. Final MFC concentrations of 10, 15, 20 and 25 μΜ are tested. The final recombinant CYP19 concentration is 7.5 nM. After incubation for 30 min at 37°C, all reactions are stopped by adding 75 μΐ of acetonitrile/0.1 M Tris base. When aromatase inhibition was tested using human placental microsomes, experimental conditions were the same as described above except that the final total protein concentration was 0.12 mg/ml. The generation of HFC is determined immediately by measuring the fluorescence response (excitation 400 nm, emission 540 nm) using a BioTek (Winooski, VT) Synergy 2 fluorometric plate reader. Standard curves are constructed using fluorescent metabolite HFC standard. Quantification of metabolite generation is carried out by applying the linear regression equation of the standard curve to the fluorescence response from each sample. The limit of quantification for HFC is 0.02 μΜ in a final volume of 200 μΐ, with intra- and inter-day coefficients of variation of 6.2% and 8.4% respectively.

Inhibition of Testosterone Metabolism by Aromatase in vitro

[0096] The activity of aromatase was determined by measuring the rate of conversion of testosterone to estradiol. All incubations were carried out using incubation times and protein concentrations that were within the linear range for reaction velocity. Testosterone and the tested inhibitors were prepared in methanol. All experiments were performed under dim light and in brown, gall tubes to minimize photodegradation of tamoxifen and its metabolites.

[0097] For reversible inhibition studies, testosterone and inhibitor were mixed at the appropriate concentrations, and methanol was removed by drying under speed vacuum before the incubation. All incubations contained recombinant human CYP19 in 100 mM sodium phosphate buffer (pH 7.4), with a NADPH-generating system (1.3 mM NADP, 3.3 mM glucose-6- phosphate, 3.3 mM MgC12, and 0.4 U/ml glucose 6-phosphate dehydrogenase) in a final volume of 250 μΐ. The reaction was prewarmed for 5 min at 37°C, initiated by the addition of the NADPH-generating system, and incubated at 37°C for 10 min. The final recombinant CYP19 concentration was 50 nM. Final testosterone concentrations of 1, 2, 4 and 8 μΜ were tested. All reactions were terminated by the addition of 20 μΐ of 60%> (w/v) perchloric acid, followed by immediate vortexing and placement of the tubes on ice. For studies designed to test for irreversible inhibition, experiments were carried out as previously described. Lu WJ, Bies R, Kamden LK, Desta Z, Flockhart DA (2010) Methadone: a substrate and mechanism-based inhibitor of CYP19 (aromatase). Drug Metab Dispos 38 (8): 1308-1313. doi: 10.1124/dmd.110.032474. The fluorometric substrate, MFC was tested under these same conditions in order to compare IC₅₀ values of tested inhibitors with a different substrate.

Quantification of Estradiol Formation

[ΘΘ98] All samples were extracted immediately after the incubations were carried out. First, 25 μΐ of 25 μΜ letrozole was added to each sample as an internal standard. The incubation mixture was then centrifuged at 14,000 rpm for 5 min at room temperature. The supernatant layer was made alkaline by adding 500 μΐ of 1 M glycine -NaOH buffer (pH 11.3) and extracted by adding 6 ml of ethyl acetate. This mixture was vortex-mixed for 10 seconds and then centrifuged at 36,000 rpm for 15 min. The organic layer was transferred to 13^x 100-mm glass culture tubes and evaporated to dryness. The resulting residue was reconstituted with mobile phase (50% 10 mM monobasic potassium phosphate, 40% acetonitrile and 10% methanol). Estradiol concentrations were analyzed immediately using high performance liquid chromatography (HPLC) assays with ultraviolet (UV) detection as previously described. The retention times of letrozole, estradiol and testosterone were approximately 3, 8, and 12 min, respectively. Peak areas for each peak were obtained from an integrator, and peak area ratios with internal standard were calculated. Standard curves were constructed by linear regression of peak area ratios. Quantification of samples was carried out by applying the linear regression equation of the standard curve to the peak area ratio. The limit of quantification for estradiol was 2.5 pmol on column, with intra- and inter-day coefficients of variation of 2.4% and 5.3% respectively.

Using aromatase inhibitors to treat test animals.

[ΘΘ99] Rats are treated with a single intraperitoneal injection of a representative aromatase inhibitor, for example, Ν,Ν-didesmethyl -4-OH-tamoxifen or vehicle control. A period of time after administering the compounds the animals are biopsied or sacrificed and tissues of the animals with high aromatase activity including ovary, brain, and adipose tissue are sampled. Aromatase activity in the samples is measured. Standard assays that can be used include following the conversion of testosterone to β-estradiol. The measured activity may be expressed as aromatase activity per unit of protein e.g., per mg of protein and/or of tissue. Aromatase activity in the treated and untreated (control) animals is compared; the level of aromatase inhibition determined maybe expressed as percent of the aromatase activity measured in animals that are dosed with the vehicle only (control). Inhibition of Aromatase using Placental Microsomes

The activity of aromatase was determined by measuring the rate of conversion of testosterone to estradiol. The incubation conditions and the quantification methods were as previously described.

Inhibition of Specific CYP450 Isoforms Using Pooled HLMs

[00100] Inhibition of individual CYP450 isoforms was studied as previously described with the modification that the formation rates of 4'-hydroxyomeprazole from omeprazole and of 6-β hydroxytestosterone from testosterone served as markers of CYP2C19 and CYP3A activity respectively. All incubations were carried out using incubation time and protein concentrations that were within the linear range for reaction velocity. All samples were extracted immediately after the incubations were carried out. The quantification methods for 6P-hydroxytestosterone formation were as previously described. Lu WJ, Ferlito V, Xu C, Flockhart DA, Caccamese S (2011) Enantiomers of naringenin as pleiotropic, stereoselective inhibitors of cytochrome P450 isoforms. Chirality. An HPLC with tandem mass spectrometry detection (LC-MS/MS) assay was developed for the quantification of the formation of R-hydroxyomeprazole. First, 25 μΐ of 1 μg/ml R-lansoprazole was added to each sample as an internal standard. The incubation mixture was then extracted by the addition of 500 μΐ of 0.025 M NaCl (pH 7.5) and 6 ml of ethyl acetate. After centrifugation at 36,000 rpm for 15 min, the organic layer was evaporated to dryness and then reconstituted in 100 μΐ of mobile phase. The separation column used was Chiral-AGP (150 x 4.60 mm; 5 μΜ; Phenomenex). A gradient elution profile was used: initial mobile phase: 95% (v/v) 20 mM ammonium acetate (adjusted to pH 6.5) and 5% acetonitrile; secondary mobile phase: 10% 20 mM ammonium acetate (adjusted to pH 6.5) and 90% acetonitrile. The secondary mobile phase was increased from 0% to 40% linearly between 0 and 8 min; the initial mobile phase was resumed after 9 min and remained constant for an additional 6 min, allowing the column to equilibrate. The elute was introduced, without splitting, at 0.5 ml/min to the turbo ion source. R-hydroxyomeprazole and R-lansoprazole were detected using multiple reactions monitoring at m/z values of 362.13/214.10 and 370.25/252.30, respectively. Formation rates of metabolites from their respective probe substrates were quantified by using the appropriate standard curve. Intra- and inter-day coefficients of variation of the assays were less than 15%. Inhibition of Recombinant Human CYP Isoforms [00101 ] The activity of each recombinant human CYP isoform was determined by measuring the conversion rate of a fluorometric substrate to its fluorescent metabolite as previously described.

Kinetic Analyses

[00102] The rates of metabolite formation from substrate probes in the presence of the test inhibitors are compared with those for control in which the inhibitor is replaced with vehicle. The extent of aromatase inhibition is expressed as percent enzyme activity remaining compared to control. The percent of aromatase activity remaining at different inhibitor concentrations is used to estimate IC₅₀ values when the substrate concentration is set at 25 μΜ. IC₅o values are determined as the inhibitor concentration that brought about a 50% reduction in enzyme activity by fitting all the data to a one-site competition equation using Prism version 5.01 for Windows (GraphPad Software Inc., San Diego, CA).

[00103] In order to estimate inhibition constants, formation rates of metabolite at different substrate concentrations are p owing equation:

wherein v is the velocity of reaction, [S] is the substrate concentration, [I] is the inhibitor concentration, K_m is the Michaelis constant, and V_max is the maximum reaction rate. The equilibrium dissociation constant of the inhibitor K_z is determined by estimating the intercept using linear regression.

[00104] To further characterize the mechanism of inhibition, the same data are plotted as Eadie-Hofstee plots according to the following equation:

wherein, Ks_app is the apparent Michaelis constant and V_ma5a is the apparent maximum reaction rate in the presence of the inhibitor. The relationships between the slopes of these lines generated by linear regression are used to determine the inhibitory mechanisms involved (24).

[00105] The same data are plotted as Lineweaver-Burk plots according to the following equation:

Using linear regression, the intercepts on the X-axis are used to determine the apparent K_m values and intercepts on the Y-axis are used to determine the apparent V_max values.

Testing the use of aromatase inhibitors as part of a method for selecting treatment options

[00106] Fresh frozen breast tumor tissue is obtained anonymously from the Indiana University School of Medicine tumor bank. The tissue samples are homogenized under conditions selected to preserve any aromatase activity present in the samples. As a control aromatase activity is measured in either the homogenates or in at least partially purified samples of the homogenates. Aromatase activity is normalized to the amount of protein in the sample analyzed, and maybe expressed in units such as aromatase activity per mg of protein. Methods for measuring aromatase activity include, for example, incubating a portion of the homogenate with testosterone, or another suitable substrate for aromatase and measuring the amount of product produced, for example, β-estradiol generated or substrate consumed.

[00107] The aromatase activity assay is repeated in the presence of an inhibitory level of at least one aromatase inhibitor, the substrate testosterone or another suitable substrate and with the homogenized tissue. Aromatase inhibitors that can be used include, for example, N,N- didesmethyl -4-OH-tamoxifen. The levels of aromatase activity measured in the presence and absence of the inhibitor are compared to one another. And the difference, if any, in aromatase activity is calculated; a statistically significant drop in aromatase activity indicates that the sample included a detectable level of at least one aromatase that can be inhibited by the type of aromatase inhibitor used in the assay.

Computerized Molecular Modeling

[00108] All tamoxifen metabolite structures were constructed with Sybyl 7.1 software and their energies minimized to 0.01 kcal/mol by the Powell method, using Gasteiger-Huckel charges and the Tripos force field. The energy-minimized structures were docked into the androgen binding pocket in aromatase after removal of the structure of the natural ligand. The parameters were set as the default values for protein-ligand docking program GOLD. The maximum distance between hydrogen bond donors and acceptors for hydrogen bonding was set to 3.5 A. After docking, the lowest-energy docking solutions of compounds of interest were merged into the ligand-free protein structure. In the case of Z-norendoxifen, the amino side chain was rotated manually to place the nitrogen atom within hydrogen bonding distance to the Ala306 carbonyl oxygen, which ultimately resulted in a more favorable calculated binding energy after energy minimization. The structures of the new ligand-protein complexes were subsequently subjected to energy minimization using the Amber force field with Amber charges. During the energy minimizations, the structures of the compounds of interest and a surrounding 10 A sphere of the protein were allowed to move. The structure of the remaining protein was kept frozen. The energy minimizations were performed using the Powell method with a 0.05 kcal/(mol A) energy gradient convergence criterion and a distance-dependent dielectric function.

[00109] While the novel technology has been illustrated and described in detail in the figures and foregoing description, the same is to be considered as illustrative and not restrictive in character, it being understood that only the preferred embodiments have been shown and described and that all changes and modifications that come within the spirit of the novel technology are desired to be protected. As well, while the novel technology was illustrated using specific examples, theoretical arguments, accounts, and illustrations, these illustrations and the accompanying discussion should by no means be interpreted as limiting the technology. All patents, patent applications, and references to texts, scientific treatises, publications, and the like referenced in this application are incorporated herein by reference in their entirety.

Claims

CLAIMS We claim:

1. A method of inhibit

contacting at least one aromatasc inhibitor of Formula A:

wherein,

Ri may be independently selected from the group consisting of H, CH3 and OH; R2 and Rj are independently selected from the group consisting of EL CH₃", CHj-CHr-

R4 is selected from the group consisting of: H, CH₃ ^", CH₃-(CH2)_n-^ hydoxy, mcthoxy, cthoxy; and

n = 1 , 2, 3, 4 or 5 and wherein Formula A inhibits at least one aromatase;

and

with at least one aromatase.

2. The method according to claim 1, wherein:

n=» 2;

Ri is independently selected from the group consisting of H, and OH;

R2 and R3 arc independently selected from the group consisting of H, or Me; and 4 is selected from the group consisting of: H, or Mc.

3. The method according to claim 1 , wherein he aromatase inhibitor is norendoxifen.

4. The method according to claim 1 , wherein the aromatasc inhibitor is endoxifen.

5. The method according to claim 1, where the contacting step occurs in vitro.

6. The method according to claim 1 , where the contacting step occurs in vivo.

7. The method according to claim 1, wherein the aromatase inhibitor is a better inhibitor of aromatase CYP19 than it is an inhibitor of the aromatases selected from the group consisting of: CYP2B6, CYP2D6, CYP2C , CYP2C19, and CYP3A.

8. A method of screening patients for treatment with aromatase inhibitors, comprising the steps of:

contacting at least one aromatase inhibitor of Formula A:

wherein,

Ri may be independently selected from the group consisting ofH, C¾ and OH; ¾ and R3 are independently selected from the group consisting of H, CH3^", CHa-CHr . CHj-CHyCHj-;

R4 is selected from the group consisting oft H, CHj^", 0¾-(Ο¼)η-, hydoxy, methoxy, ethoxy, with a sample, wherein said sample is a biological fluid or tissue; and

n = 1 , 2, 3, 4 or 5 and wherein Formula A inhibits at least one aromatase

measuring the level of aromatase activity in bom the presence and in absence of said aromatase inhibitor Formula A in a sample of tissue, blood, cells and/or Quid from a patient; and

assigning a patient whose sample demonstrates a large change in aromatase activity measured in the presence and in the absence of said aromatase inhibitor to a group that is disposed to treatment with an aromatase inhibitor.

9. The method according to claim 8, wherein the aromatase inhibitor is selected from the group consisting of endoxifen and norendoxifen or a pharmaceutically acceptable salt mercof.

10. A method of treating a patient, comprising the steps of:

identifying a patient in need of an aromatase inhibitor; and

administering a therapeutically effective amount of a compound according to Formula A, or a pharmaceutical ry acceptable salt mercof:

wherein,

Ri may be independently selected from the group consisting of H, CH3 and OH; Rs and R3 arc independently selected from the group consisting of H, CH3^", CH3-CH2- , CH₃-CH₂-CH₂-;

R is selected from the group consisting of: H, CHj^", <¾-{<¾),,-. hydoxy, methoxy, ethoxy, and

n = 1, 2, 3, 4 or 5 or a pharmaceutically acceptable salt thereof.

11. The method according to claim 10, wherein

n = 2;

Ri is independently selected from the group consisting of H, and OH;

R₂ and R3 arc independently selected from the group consisting of H, or Me; and

R4 is selected from the group consisting of: H, or Me.

12. The method according to claim 10 wherein the aromatase inhibitor is norendoxifen or a pharmaceutically acceptable salt thereof

13. The method according to claim 10, wherein the aromatase inhibitor is endoxifen or a pharmaceutically acceptable salt thereof.

14. The method according to claim 10, wherein the patient is an animal.

15. The method according to claim 10, wherein the patient is a human being.

16. The method according to claim 10, fuiliwmcludingthe step of:

monitoring the course of the diseasing by obtaining at least one more sample of tissue, cells, blood or fluid from the patient after said patient is treated with at least one therapeutic dose of the compound for Formula A.

17. The method according to claim 10, wherein the patient, treated with at least one . compound of Formula A is also treated with at least one other compound.

18. The method according to claim 10, wherein the patient treated with at least ne compound of Formula A is also treated with at least one other therany en tmt ⁽mm «*» group consisting of: radiation, convention chemotherapy and surgery.

19. The method according to claim 10, wherein the patient is symptomatic for breast cancer.

20. The method according to claim 10, wherein the aromatase inhibitor is a better inhibitor of aromatase CYP19 man it is an inhibitor of the sromatases selected from the group consisting of: CYP2B6, CYP2D6, CYP2C9, CYP2C19, and CYP3A.

21. An aromatase inhibitor comprising: an aromatase inhibitor of Formula A:

wherein,

Ri may be independently selected from the group consisting of H, CH3 and OH; R2 and R3 arc independently selected from the group consisting of H, CH3^", CH3-CH2- , CH3-CH2-CH2-;

R4 is selected from the group consisting of:

hydoxy, mcthoxy, ethoxy; and

n = I, 2, 3, 4 or 5 and wherem Fonnula A mhibits at 1(^ one aromatase.

22. The aromatase inhibitor according to claim 22, wherein:

n = 2;

Ri is independently selected from die group consisting of H, and OH;

R2 and Rj are independently selected from the group consisting of H, or Me; and

R4 is selected from the group consisting of: H, or Me.

23. The aromatase inhibitor according to claim 22, wherein said aromatase inhibitor is a better inhibitor of aromatase CYP19 than it is an inhibitor of the aromatases selected from the group consisting of: CYP286, CYP2D6, CYP2C9, CYP2C19, and CYP3A.