EP1343803A2 - 2-substituierte estrogene mit antiangiogenischer aktivität - Google Patents

2-substituierte estrogene mit antiangiogenischer aktivität

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Publication number
EP1343803A2
EP1343803A2 EP01968112A EP01968112A EP1343803A2 EP 1343803 A2 EP1343803 A2 EP 1343803A2 EP 01968112 A EP01968112 A EP 01968112A EP 01968112 A EP01968112 A EP 01968112A EP 1343803 A2 EP1343803 A2 EP 1343803A2
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Prior art keywords
compound
absent
coh
chch
alkyl
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French (fr)
Inventor
Gregory Agoston
Jamshed H. Shah
Kimberly A. Hunsucker
Victor Pribluda
Theresa M. Lavallee
Shawn J. Green
Christopher J. Herbstritt
Xiaoguo H. Zhan
Anthony Treston
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Casi Pharmaceuticals Inc
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Entremed Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J1/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, not substituted in position 17 beta by a carbon atom, e.g. estrane, androstane
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present invention relates to treating disease states characterized by abnormal cell mitosis and to treating disease states characterized by abnormal angiogenesis and to treating disease states characterized by a combination of these events. More particularly, the present invention relates to analogs of 2-methoxyestradiol (2ME ) and their effect on diseases characterized by abnormal cell mitosis and/or abnormal angiogenesis.
  • 2ME 2-methoxyestradiol
  • Angiogenesis is the generation of new blood vessels into a tissue or organ. Under normal physiological conditions, humans and animals undergo angiogenesis only in very specific, restricted situations. For example, angiogenesis is normally observed in wound healing, fetal and embryonal development, and formation of the corpus luteum, endometrium and placenta.
  • Angiogenesis is controlled through a highly regulated system of angiogenic stimulators and inhibitors.
  • the control of angiogenesis has been found to be altered in certain disease states and, in many cases, pathological damage associated with the diseases is related to uncontrolled angiogenesis. Both controlled and uncontrolled angiogenesis are thought to proceed in a similar manner.
  • Endothelial cells and pericytes surrounded by a basement membrane, form capillary blood vessels.
  • Angiogenesis begins with the erosion of the basement membrane by enzymes released by endothelial cells and leukocytes. Endothelial cells, lining the lumen of blood vessels, then protrude through the basement membrane.
  • Angiogenic stimulants induce the endothelial cells to migrate through the eroded basement membrane.
  • the migrating cells fonn a "sprout" off the parent blood vessel where the endothelial cells undergo mitosis and proliferate.
  • the endothelial sprouts merge with each other to form capillary loops, creating a new blood vessel.
  • Persistent, unregulated angiogenesis occurs in many disease states, tumor metastases, and abnormal growth by endothelial cells.
  • the diverse pathological disease states in which unregulated angiogenesis is present have been grouped together as angiogenic-dependent or angiogenic-associated diseases.
  • ocular neovascular disease This disease is characterized by invasion of new blood vessels into the structures of the eye, such as the retina or cornea. It is the most common cause of blindness and is involved in approximately twenty eye diseases. In age-related macular degeneration, the associated visual problems are caused by an ingrowth of choroidal capillaries through defects in Bruch's membrane with proliferation of fibrovascular tissue beneath the retinal pigment epithelium. Angiogenic damage is -also associated with diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, neo vascular glaucoma, and retrolental fibroplasia.
  • corneal neovascularization include, but are not limited to, epidemic keratoconjunctivitis, Nitamin A deficiency, contact lens overwear, atopic keratitis, superior limbic keratitis, and pterygium keratitis sicca.
  • Other diseases associated with undesirable angiogenesis include Sj ⁇ gren's syndrome, acne rosacea, phylectenulosis, syphilis, Mycobacteria infections, lipid degeneration, chemical burns, bacterial ulcers, fungal ulcers, Herpes simplex infection, Herpes zoster infections, protozoan infections, Kaposi's sarcoma, Mooren's ulcer, Terrien's marginal degeneration, marginal keratolysis, rheumatoid arthritis, systemic lupus, polyarteritis, trauma, Wegener's sarcoidosis, scleritis, Stevens- Johnson's disease, pemphigoid, and radial keratotomy.
  • Diseases associated with retinal/choroidal neovascularization include, but are not limited to, diabetic retinopathy, macular degeneration, sickle cell anemia, sarcoidosis, syphilis, pseudoxanthoma elasticum, Paget's disease, vein occlusion, artery occlusion, carotid obstructive disease, chronic uveitis/vitritis, Mycobacteria infections, lyme's disease, systemic lupus erythematosis, retinopathy of prematurity, Eales' disease, Behcet's disease, infections causing retinitis or choroiditis, presumed ocular histoplasmosis.
  • Eye-related diseases include, but are not limited to, diseases associated with rubeosis (neovascularization of the angle) and diseases caused by the abnormal proliferation of fibrovascular or fibrous tissue, including all forms of prolific vitreoretinopathy.
  • angiogenesis associated disease is rheumatoid arthritis.
  • the blood vessels in the synovial lining of the joints undergo angiogenesis.
  • the endothelial cells release factors and reactive oxygen species that lead to pannus growth and cartilage destruction.
  • Angiogenesis may also play a role in osteoarthritis.
  • the activation of the chondrocytes by angiogenic-related factors contributes to the destruction of the joint.
  • the angiogenic factors promote new bone growth.
  • Therapeutic intervention that prevents the bone destruction could halt the progress of the disease and provide relief for persons suffering with arthritis.
  • Chronic inflammation may also involve pathological angiogenesis.
  • pathological angiogenesis Such diseases as ulcerative colitis and Crohn's disease show histological changes with the ingrowth of new blood vessels and the inflamed tissues. Bartonelosis, a bacterial infection found in South America, can result in a chronic stage that is characterized by proliferation of vascular endothelial cells.
  • Another pathological role associated with angiogenesis is found in atherosclerosis. The plaques formed within the lumen of blood vessels have been shown to have angiogenic stimulatory activity.
  • Tumor 'take' has occurred, every increase in tumor cell population must be preceded by an increase in new capillaries converging on the tumor.
  • Tumor 'take' is currently understood to indicate a prevascular phase of tumor growth in which a population of tumor cells occupying a few cubic millimeters volume, and not exceeding a few million cells, can survive on existing host microvessels. Expansion of tumor volume beyond this phase requires the induction of new capillary blood vessels. For example, pulmonary micrometastases in the early prevascular phase in mice would be undetectable except by high power microscopy on histological sections.
  • Tumor growth in the avascular cornea proceeds slowly and at a linear rate, but switches to exponential growth after neovascularization.
  • Tumors suspended in the aqueous fluid of the anterior chamber of the rabbit eye remain viable, avascular, and limited in size to ⁇ 1 mm 3 . Once they are implanted on the iris vascular bed, they become neovascularized and grow rapidly, reaching 16,000 times their original volume within 2 weeks. (Gimbrone, Jr., et al., J. Exp. Med., 136:261-76).
  • Vascular casts of metastases in the rabbit liver reveal heterogeneity in size of the metastases, but show a relatively uniform cut-off point for the size at which vascularization is present.
  • Tumors are generally avascular up to 1 mm in diameter, but are neovascularized beyond that diameter. (Lien, et al, Surgery, 68:334-40 (1970)).
  • pre-vascular hyperplastic islets are limited in size to ⁇ 1 mm.
  • 4- 10% of the islets become neovascularized, and from these islets arise large vascularized tumors of more than 1000 times the volume of the pre-vascular islets.
  • VEGF vascular endothelial growth factor
  • Anti-bFGF monoclonal antibody causes 70% inhibition of growth of a mouse tumor which is dependent upon secretion of bFGF as its only mediator of angiogenesis. The antibody does not inhibit growth of the tumor cells in vitro. (Hori, et al, Cancer Res., 51:6180-84 (1991)).
  • bFGF Intraperitoneal injection of bFGF enhances growth of a primary tumor and its metastases by stimulating growth of capillary endothelial cells in the tumor.
  • the tumor cells themselves lack receptors for bFGF, and bFGF is not a mitogen for the tumors cells in vitro. (Gross, et al, Proc. Am. Assoc. Cancer Res., 31:79 (1990)).
  • a specific angiogenesis inhibitor (AGM-1470) inhibits tumor growth and metastases in vivo, but is much less active in inhibiting tumor cell proliferation in vitro. It inhibits vascular endothelial cell proliferation half-maximally at 4 logs lower concentration than it inhibits tumor cell proliferation. (Ingber, et al, Nature, 48:555-57 (1990)). There is also indirect clinical evidence that tumor growth is angiogenesis dependent.
  • angiogenesis plays a major role in the metastasis of cancer. If this angiogenic activity could be repressed or eliminated, then the tumor, although present, would not grow. In the disease state, prevention of angiogenesis could avert the damage caused by the invasion of the new ' micro vascular system. Therapies directed at control of the angiogenic processes could lead to the abrogation or mitigation of these diseases.
  • Angiogenesis has been associated with a number of different types of cancer, including solid tumors and blood-borne tumors.
  • Solid tumors with which angiogenesis has been associated include, but are not limited to, rhabdomyosarcomas, retinoblastoma, Ewing's sarcoma, neuroblastoma, and osteosarcoma.
  • Angiogenesis is also associated with blood-borne tumors, such as leukemias, any of various acute or chronic neoplastic diseases of the bone marrow in which unrestrained proliferation of white blood cells occurs, usually accompanied by anemia, impaired blood clotting, and enlargement of the lymph nodes, liver and spleen. It is believed to that angiogenesis plays a role in the abnormalities in the bone marrow that give rise to leukemia tumors and multiple myeloma diseases.
  • hemangioma One of the most frequent angiogenic diseases of childhood is the hemangioma.
  • a hemangioma is a tumor composed of newly-formed blood vessels. In most cases the tumors are benign and regress without intervention. In more severe cases, the rumors progress to large cavernous and infiltrative forms and create clinical complications. Systemic forms of hemangiomas, hemangiomatoses, have a high mortality rate. Therapy-resistant hemangiomas exist that cannot be treated with therapeutics currently in use.
  • Angiogenesis is also responsible for damage found in heredity diseases such as Osler- Weber-Rendu disease, or heredity hemorrhagic telangiectasia. This is an inherited disease characterized by multiple small angiomas, tumors of blood or lymph vessels. The angiomas are found in the skin and mucous membranes, often accompanied by epitaxis (nose bleeds) or gastrointestinal bleeding and sometimes with pulmonary or hepatitic arteriovenous fistula.
  • composition and method which can inhibit angiogenesis.
  • composition and method which can inhibit the unwanted growth of blood vessels, especially in tumors.
  • Angiogenesis is also involved in normal physiological processes, such as reproduction and wound healing. Angiogenesis is an important step in ovulation and also in implantation of the blastula after fertilization. Prevention of angiogenesis could be used to induce amenorrhea, to block ovulation, or to prevent implantation by the blastula.
  • Interferon a 4 kDa glycoprotein from bovine vitreous humor and a cartilage derived factor
  • a 4 kDa glycoprotein from bovine vitreous humor and a cartilage derived factor have been used to inhibit angiogenesis.
  • Cellular factors such as interferon, inhibit angiogenesis.
  • interferon alpha or human interferon beta have been shown to inhibit tumor-induced angiogenesis in mouse dermis stimulated by human neoplastic cells.
  • Interferon beta is also a potent inhibitor of angiogenesis induced by allogeneic spleen cells. (Sidky, et al, Cancer Res., 47:5155- 61(1987)).
  • Thalidomide is a hypnosedative that has been successfully used to treat a number of angiogenesis-associated diseases, such as rheumatoid arthritis (Gutierrez-Rodriguez, Arthritis Rheum., 27 (10):1118-21 (1984); Gutierrez-Rodriguez, et al, J. Rheumatol, 16(2): 158-63 (1989)), Behcet's disease (Handley, et al, Br. J.
  • thalidomide Although thalidomide has minimal side effects in adults, it is a potent teratogen. Thus, there are concerns regarding its use in women of child-bearing age. Although minimal, there are a number of side effects which limit the desirability of thalidomide as a treatment. One such side effect is drowsiness. In a number of therapeutic studies, the initial dosage of thalidomide had to be reduced because patients became lethargic and had difficulty functioning normally. Another side effect limiting the use of thalidomide is peripheral neuropathy, in which individuals suffer from numbness and disfunction in their extremities.
  • 2-Methoxyestradiol is an endogenous metabolite of estradiol (E 2 ) that has potent anti- proliferative activity and induces apoptosis in a wide variety of tumor and non-tumor cell lines.
  • the present invention provides certain analogs of 2-methoxyestradiol that are effective in treating diseases characterized by abnormal mitosis and/or abnormal angiogenesis. Specifically the present invention relates to analogs of 2-methoxyestradiol that have been modified at the 2, 16 or 17 positions or combinations thereof. Compounds within the general formulae that inhibit cell proliferation are preferred. Compounds within the general formula that inhibit angiogenesis are also preferred. Preferred compositions may also exhibit a change (increase or decrease) in estrogen receptor binding, improved absorption, transport (e.g. through blood-brain barrier and cellular membranes), biological stability, or decreased toxicity. The invention also provides compounds useful in the method, as described by the general formulae of the claims.
  • a mammalian disease characterized by undesirable cell mitosis includes but is not limited to excessive or abnormal stimulation of endothelial cells (e.g., atherosclerosis), solid tumors and tumor metastasis, benign tumors, for example, hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas, vascular malfunctions, abnormal wound healing, inflammatory and immune disorders, Bechet's disease, gout or gouty arthritis, abnormal angiogenesis accompanying: rheumatoid arthritis, skin diseases, such as psoriasis, diabetic retinopathy and other ocular angiogenic diseases such as retinopathy of prematurity (retrolental fibroplasic), macular degeneration, corneal graft rejection, neovascular glaucoma and Osier Weber syndrome (Osier- Weber-Rendu disease).
  • endothelial cells e.g., atherosclerosis
  • compositions described above can be used to block ovulation and implantation of a blastula or to block menstruation (induce amenorrhea).
  • 2-methoxyestradiol an endogenous metabolite of estradiol with no intrinsic estrogenic activity, is a potent antiproliferative agent that induces apoptosis in a wide variety of tumor and non-tumor cell lines. When administered orally, it exhibits antitumor and antiangiogenic activity with little or no toxicity.
  • 2ME 2 is in several ⁇ hase-I and II clinical trials under the name PANZEM TM.
  • a novel series of compounds have been prepared that retain the biological activities of 2ME 2 but are expected to have reduced metabolism.
  • Several analogs lack the hydroxyl moiety at position 17 and cannot be metabolized to 2-methoxyestrone or conjugated at that position.
  • Another set of compounds have the 2-methoxy group replaced by moieties which cannot be de-methylated to yield "the potentially-estrogenic 2-hydroxy derivatives.
  • Contrary to what is observed with 2ME 2 several of these new analogs have selective in vitro antiproliferative activity for the endothelial cells over the tumor cell line assessed. The synthesis and SAR properties of these potential antitumor and antiangiogenic compounds will be discussed.
  • Position alkylated analogs lack the hydroxyl moiety and cannot be metabolized to 2- methoxyestrone or conjugated at that position but retain antiproliferative activity in HUVEC and MDA-MB-231 cells.
  • compounds that are useful in accordance with the invention include novel 2-methoxyestradiol derivatives that exhibit anti-mitotic, anti-angiogenic and anti-tumor properties.
  • Specific compounds according to the invention are described below.
  • Preferred compounds of the invention are 2-methoxyestradiol derivatives modified at the 2, 16, or 17 positions or combinations thereof.
  • the invention extends to other compounds within the formulae given in the claims below, having the described characteristics. These characteristics can be determined for each test compound using the assays detailed below and elsewhere in the literature.
  • 2-Methoxyestradiol is an endogenous metabolite of estradiol that has potent antiproliferative activity and induces apoptosis in a wide variety of tumor and non-tumor cell lines. When administered orally, it exhibits anti-tumor and anti-proliferative activity with little or no toxicity.
  • 2-Methoxyestradiol is metabolized to a less active metabolite, 2-methoxyestrone (2ME ⁇ ) as indicated by in vitro and in vivo results. Although not wishing to be bound by theory, it is believed that this metabolite is formed through the same enzymatic pathway as estrone is formed from estradiol.
  • estradiol the enzymes responsible for this reaction on estradiol are the 17 ⁇ -hydroxysteroid dehydrogenases (17 ⁇ -HSD) which utilize NADP+ as a co-factor (Han et al, J. Biol Chem. 275:2, 1105-1111 (Jan. 12, 2000) and other references cited earlier).
  • 17 ⁇ -HSD 17 ⁇ -hydroxysteroid dehydrogenases
  • types 1, 2, 3, and 4 have distinct activity. It appears that 17 ⁇ - HSD type 1 catalyzes the reductive reaction (estrone to estradiol), while 17 ⁇ -HSD type 2 catalyzes the oxidation reaction (estradiol to estrone), and type 3 catalyzes 4-androstenedione to testosterone.
  • positions 16 and/or 17 of 2-methoxyestradiol may be modified to prevent these metabolic pathways from occurring.
  • the present invention adds steric bulk and/or modification of chemical or electrostatic characteristics at positions 16 and 17 of 2-methoxyestradiol for retarding or preventing interaction of the family of 17 ⁇ -hydroxy steroid dehydrogenases and co-factor NADP + on this substrate. Addition of steric bulk and/or modification of chemical or electrostatic characteristics at positions 16 and 17 of 2-methoxyestradiol may also retard or prevent conjugation, such as glucuronidation.
  • estradiol (E 2 ) and ethynyl-E 2 are extensively metabolized during passage through the gastrointestinal tract and by first-pass metabolism in the liver.
  • estradiol (E 2 ) and ethynyl-E 2 are extensively metabolized during passage through the gastrointestinal tract and by first-pass metabolism in the liver.
  • Two major metabolic pathways that lead to rapid deactivation and excretion are well studied (Fotsis, T.; Zhang, Y.; Pepper, M. S.; Adlercrcutz, H.; Montesano,
  • derivatives are modified at combinations of the 2, 16 or 17 positions.
  • Anti-Proliferative activity is evaluated in situ by testing the ability of an improved estradiol derivative to inhibit the proliferation of new blood vessel cells (angiogenesis).
  • a suitable assay is the chick embryo chorioallantoic membrane (CAM) assay described by Crum et al. Science 230:1375 (1985). See also, U.S. Patent 5,001,116, hereby incorporated by reference, which describes the CAM assay. Briefly, fertilized chick embryos are removed from their shell on day 3 or 4, and a methylcellulose disc containing the drug is implanted on the chorioallantoic membrane.
  • the embryos are examined 48 hours later and, if a clear avascular zone appears .around the methylcellulose disc, the diameter of that zone is measured. Using this assay, a 100 ⁇ g disk of the estradiol derivative 2-methoxyestradiol was found to inhibit cell mitosis and the growth of new blood vessels after 48 hours. This result indicates that the anti-mitotic action of 2-methoxyestradiol can inhibit cell mitosis and angiogenesis.
  • 2ME 2 induced changes in the levels and activities of various proteins involved in the progression of the cell cycle. These include cofactors of DNA replication and repair, e.g., proliferating cell nuclear antigen (PCNA) (Klauber, N., Parangi, S., Flynn, E., Hamel, E. and D'Amato, R.J. (1997), Inhibition of angiogenesis and breast cancer in mice by the microtubule inhibitors 2- methoxyestradiol and Taxol., Cancer Research 57, 81-86; Lottering, M-L., de Kock, M.,
  • PCNA proliferating cell nuclear antigen
  • 2-Methoxyestradiol an- endogenous estrogen metabolite induces apoptosis in endothelial cells and inhibits angiogenesis: Possible role for stress-activated protein kinase signaling pathway and fas expression.
  • tubulin D'Amato, R.J., Lin, CM., Flynn, E., Folkman, J. and Hamel, E. (1994) 2-Methoxyestradiol, and endogenous mammalian metabolite, inhibits tubulin polymerization by interacting at the colchicine site. Proc. Natl. Acad. Sci. USA 91, 3964-3968; Hamel, E., Lin, CM., Flynn, E. and D'Amato, R.J.
  • 2-Methoxyestradiol induces apoptosis throughactivation of the extrinsic pathway. (Manuscript in preparation)). Additionally, 2ME2 has been shown to interact with superoxide dismutase (SOD) 1 and SOD 2 and to inhibit their enzymatic activities (Huang, P., Feng, L., Oldham, E. A., Keating, M. J., and Plunkett, W. 2000. Superoxide dismutase as a target for the selective killing of cancer cells, Nature. 407:390-5.).
  • SOD superoxide dismutase
  • the high affinity binding to SHBG has been mechanistically associated to its efficacy in a canine model of prostate cancer, in which signaling by estradiol and 5 ⁇ -androstan-3 ,17 ⁇ - diol were inhibited by 2ME 2 (Ding, V.D., Moller, D.E., Feeney, W.P., Didolkar, V., Nakhla,
  • anti-mitotic activity mediated by effects on tubulin polymerization activity can be evaluated by testing the ability of an estradiol derivative to inhibit tubulin polymerization and microtubule assembly in vitro. Microtubule assembly is followed in a
  • a reaction mixture typically contains 1.0M monosodium ' glutamate (pH 6.6), 1.0 mg/ml (lO ⁇ M) tubulin, 1.0 mM MgC-2, 4% (v/v) dimethylsulfoxide and 20-
  • reaction mixtures 75 ⁇ M of a composition to be tested.
  • the reaction mixtures are incubated for 15 min. at 37°C and then chilled on ice. After addition of lO ⁇ l 2.5mM GTP, the reaction mixture is transferred to a cuvette at 0°C, and a baseline established. At time zero, the temperature controller of the spectrophotometer is set at 37°C Microtubule assembly is evaluated by increased turbity at 350 mn. Alternatively, inhibition of microtubule assembly can be followed by transmission electron microscopy as described in Example 2 of U.S. Patent Nos. 5,504,074, 5,661,143, and 5,892,069.
  • antiangiogenic activity may be evaluated through endothelial cell migration, endothelial cell tubule formation, or vessel outgrowth in ex-vivo models such as rat aortic rings.
  • the invention can be used to treat any disease characterized by abnormal cell mitosis.
  • diseases include, but are not limited to: abnormal stimulation of endothelial cells (e.g., atherosclerosis), solid tumors and tumor metastasis, benign tumors, for example, hemangiomas, acoustic neuromas, neurofribomas, trachomas, and pyogenic granulomas, vascular malfunctions, abnormal wound healing, inflammatory and immune disorders, Bechet's disease, gout or gouty arthritis, abnormal angiogenesis accompanying: rheumatoid arthritis, skin diseases, such as psoriasis, diabetic retinopathy, and other ocular angiogenic diseases such as retinopathy of prematurity (retrolental fibroplasic), macular degeneration, corneal graft rejection, neuroscular glaucoma, liver diseases and Oster Webber syndrome (Osier- Weber Rendu disease).
  • endothelial cells e.
  • corneal neovascularization Diseases associated with corneal neovascularization that can be treated according to the present invention include but are not limited to, diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, neo vascular glaucoma and retrolental ibroplasias, epidemic keratoconjunctivitis, Vitamin A deficiency, contact lens overwear, atopic keratitis, superior limbic keratitis, pterygium keratitis sicca, sjogrens, acne, rosacea, phylectenulosis, syphilis, Mycobacteria " infections, lipid degeneration, chemical burns, bacterial ulcers, fungal ulcers, Herpes simplex infections, Herpes zoster infections, protozoan infections, Kaposi's sarcoma, Mooren's ulcer, Terrien's marginal degeneration, mariginal keratolysis, trauma, rhe
  • Diseases associated with retinal/choroidal neovascularization that can be treated according to the present invention include, but are not limited to, diabetic retinopathy, macular degeneration, sickle cell anemia, sarcoid, syphilis, pseudoxanthoma elasticum, Paget's disease, vein occlusion, artery occlusion, carotid obstructive disease, chronic uveitis/vitritis, mycobacterial infections, Lyme's disease, systemic lupus erythematosis, retinopathy of prematurity, Eales' disease, Behcet's disease, infections causing a retinitis or choroiditis, presumed ocular histoplasmosis, Best's disease, myopia, optic pits, Stargart's disease, pars planitis, chronic retinal detachment, hyperviscosity syndromes, toxoplasmosis, trauma and post-laser complications.
  • diseases include, but are not limited to, diseases associated with rubeosis (neovasculariation of the angle) and diseases caused by the abnormal proliferation of fibrovascular or fibrous tissue including all forms of proliferative vitreoretinopathy, whether or not associated with diabetes.
  • Another disease which can be treated according to the present invention is rheumatoid arthritis. It is believed that the blood vessels in the synovial lining of the joints undergo angiogenesis. In addition to forming new vascular networks, the endothelial cells release factors and reactive oxygen species that lead to pannus growth and cartilage destruction. The factors involved in angiogenesis may actively contribute to, and help maintain, the chronically inflamed state of rheumatoid arthritis.
  • Another disease that can be treated according to the present invention are hemangiomas, Osier- Weber-Rendu disease, or hereditary hemorrhagic telangiectasia, solid or blood borne tumors and acquired immune deficiency syndrome.
  • the invention can be used to treat a variety of post-menopausal symptoms, osteoporosis, cardiovascular disease, Alzheimer's disease, to reduce the incidence of strokes, and as an alternative to prior estrogen replacement therapies.
  • the compounds of the present invention can work by estrogenic and non-estrogenic biochemical pathways.
  • the present invention also relates to conjugated prodrugs and uses thereof. More particularly, the invention relates to conjugates of estradiol compounds such as
  • 2-methoxyestradiol and functionally active analogues and derivatives thereof and the use of such conjugates in the prophylaxis or treatment of conditions associated with enhanced angiogenesis or accelerated cell division, such as cancer, and inflammatory conditions such as asthma and rheumatoid arthritis and hyperproliferative skin disorders including psoriasis.
  • the invention also relates to compositions including the prodrugs of the present invention and methods of synthesizing the prodrugs.
  • the present invention provides a conjugated prodrug of an estradiol compound, preferably of 2-methoxyestradiol or a functionally active analogue or derivative thereof, conjugated to a biological activity modifying agent.
  • analogue or derivative of 2-methoxyestradiol has one or more of the biological activities of 2-methoxyestradiol.
  • the biological activities of 2-methoxyestradiol include, but are not limited to: inhibition of endothelial cell proliferation; inhibition of smooth muscle cell proliferation; inhibition of tumour cell proliferation inhibition of microtubule function; inhibition of leukocyte activation.
  • Examples of such functionally active analogues or derivatives include 2-ethoxyestradiol, 2-hydroxyestradiol and other analogues modified at the 2 position, 2-methoxyestradiol-3-methylether, 4-methoxyestradiol, and other analogues in which the B ring is expanded to a 7-numbered ring. See also W095/04535 and WO 01/27132 the entire disclosures of which are incorporated herein by reference.
  • the conjugated prodrug according to the present invention includes 2-methoxyestradiol or a functionally active analogue or derivative thereof, conjugated to a peptide moiety.
  • estradiol compound such as 2-methoxyestradiol
  • the pharmaceutical composition of this invention may also contain, or be co-administered (simultaneously or sequentially) with, one or more pharmacological agents of value in treating one or more disease conditions referred to hereinabove.
  • the prodrug may be incorporated into biodegradable polymers allowing for sustained release, the polymers being implanted in the vicinity of where delivery is desired, for example, at the site of a tumour.
  • biodegradable polymers and their use are described in detail in Brem et al., J. Neurosurg 74:441-446 (1991). A person skilled in the art will be able by reference to standard texts, such as
  • a conjugated prodrug according to the present invention for the preparation of a medicament for the prophylaxis or treatment of conditions associated with angiogenesis or accelerated cell division or inflammation.
  • a pharmaceutical composition comprising a conjugated prodrug according to the present invention, together with a pharmaceutically acceptable carrier, diluent or excipient.
  • the pharmaceutical composition may be used for the prophylaxis or treatment of conditions associated with angiogenesis or accelerated cell division or inflammation.
  • a method of prophylaxis or treatment of a condition associated with angiogenesis or accelerated or increased amounts of cell division hypertrophic growth or inflammation including administering to a patient in need of such prophylaxis or treatment an effective amount of a conjugated prodrug according to the present invention, as described above.
  • prophylaxis or treatment of said condition includes amelioration of said condition.
  • an effective amount is meant a therapeutically or prophylactically effective amount. Such amounts can be readily determined by an appropriately skilled person, taking into account the condition to be treated, the route of administration and other relevant factors. Such a person will readily be able to determine a suitable dose, mode and frequency of administration.
  • Pharmaceutically acceptable salts of the compound of the formula may be prepared in any conventional manner for example from the free base and acid. In vivo hydrolysable esters, amides and carbamates may be prepared in any conventional manner.
  • estradiol The chemical synthesis of estradiol has been described (Eder, V. et al., Ber 109, 2948 (1976); Oppolzer, D.A. and Roberts, DA. Helv. Chim. Acta. 63, 1703, (1980)).
  • the synthetic pathways used to prepare some of the derivatives of the present invention are based on modified published literature procedures for estradiol derivatives and dimethylhydrazone (Trembley et al., Bioorganic & Med. Chem. 1995 3, 505-523; Fevig et al., J. Org.
  • compositions described above can be provided as physiologically acceptable formulations using known techniques, and these formulations can be administered by standard routes.
  • the combinations may be administered by the topical, oral, rectal or parenteral (e.g., intravenous, subcutaneous or intramuscular) route.
  • the combinations may be incorporated into biodegradable polymers allowing for sustained release, the polymers being implanted in the vicinity of where delivery is desired, for example, at the site of a tumor or within or near the eye.
  • biodegradable polymers and their use are described in detail in Brem et al., J. Neurosurg. 74:441-446 (1991).
  • the dosage of the composition will depend on the condition being treated, the particular derivative used, and other clinical factors such as weight and condition of the patient and the route of administration of the compound. However, for oral administration to humans, a dosage of 0.01 to 100 mg/kg/day, preferably 0.01-20 mg/kg/day, is generally sufficient.
  • the formulations include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intraocular, intratracheal, and epidural) administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by conventional pharmaceutical techniques. Such techniques include the step of bringing into association the active ingredient and the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into associate the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous, liquid; or as an oil-in- water liquid emulsion or a water-in-oil emulsion and as a bolus, etc.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing, in a suitable machine, the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface-active or dispersing agent.
  • Molded tablets may be made by molding, in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets may optionally be coated or scored and may be formulated so as to provide a slow or controlled release of the active ingredient therein.
  • Formulations suitable for topical administration in the mouth include lozenges comprising the ingredients in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the ingredient to be administered in a suitable liquid carrier.
  • Formulations suitable for topical administration to the skin may be presented as ointments, creams, gels and pastes comprising the ingredient to be administered in a pharmaceutical acceptable carrier.
  • a preferred topical delivery system is a transdermal patch containing the ingredient to be administered.
  • Formulations for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate.
  • Formulations suitable for nasal administration include a coarse powder having a particle size, for example, in the range of 20 to 500 microns which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose.
  • Suitable formulations, wherein the carrier is a liquid, for administration, as for example, a nasal spray or as nasal drops, include aqueous or oily solutions of the active ingredient.
  • Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such as carriers as are known in the art to be appropriate.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) conditions requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tables of the kind previously described.
  • Preferred unit dosage formulations are those containing a daily dose or unit, daily sub- dose, as herein above recited, or an appropriate fraction thereof, of the administered ingredient. It should be understood that in addition to the ingredients, particularly mentioned above, the formulations of the present invention may include other agents conventional in the art having regard to the type of formulation in question, for example, those suitable for oral administration may include flavoring agents.
  • 2-Methoxyestradiol is an endogenous metabolite of estradiol (E 2 ) that has potent antiproliferative activity and induces apoptosis in a wide variety of tumor and non-tumor cell lines. When administered orally, it exhibits anti-tumor and anti-proliferative activity with little or no toxicity.
  • E 2 estradiol
  • 2-methoxyestradiol does not engage the estrogen receptor for its anti-proliferative activity.
  • the presence of demethylases in vivo may metabolize this compound to 2-hydroxyestradiol, which has been shown to be estrogenic by several approaches.
  • the present invention improves the bioavailability of estradiol or 2- methoxyestradiol and reduces the formation of estrogenic 2-methoxyestradiol metabolities.
  • the present invention modifies estradiol analogs in such a way to prevent or hinder demethylation, oxidation, and conjugation with another molecule during metabolism.
  • the present invention includes compositions and methods for treating mammalian disease characterized by pathogenic angiogenesis by administering derivates of 2- methoxyestradiol.
  • the derivatives may be modified at the 2, 16, or 17 positions or combinations thereof, where it is chemically possible to someone skilled in the art. Combinations which are physically impossible are not contemplated by this invention, such as a carbon atom containing 5 bonds.
  • a hetero groups is defined herein as any group which contains at least one atom that is not C or H.
  • a hetero group may contain other substituents, such as aromatic rings and other functional groups.
  • the hetero group may be directly attached to the ring or on a substituent of a group. Especially considered are O, N, S, and P.
  • positions 2, 16 and 17 are the modifications of acid, amide, amine, linear and branched chain alkanes, alkenes and alkynes with heteroatom substitutions, including, but not limited to, carbonyl, -CO-, -S-, -NH-, and or -O- instead of CH 2 and also optionally substituted with hydroxyl, amino, sulphydryl, azide, halides, nitro, azides, nitrile, sulfamate, carbamate, phosphate, azides and azos, ester, ether, halide, formamide, nitro, nitrile, sulfide, sulfoxide, sulfate, sulfamate, phosphate, and phosphonate instead of H; single or multiple homocyclic or heterocyclic rings of 3, 4, 5, 6, 7 or 8 members, either saturated or unsaturated, attached directly to the 2, 16 or 17 position or linked via linear or branched chain alkanes, alkene
  • R is hydrogen; ii) R is alkyl chains, straight and branched with stereoisomers up to IOC; iii) R is alkene or alkyne derivatives of above alkyl chain with the olefin or alkyne moiety at any position and any configuration on the chain. Also included are multiply unsaturated alkyl chains of any configuration up to 10.
  • the alkyl chain could be substituted with a phenyl substitutent and substituted phenyl substiutents (examples include, but are not limited to, aniline, anisole, toluene, phenol); iv) alkyl, alkene or alkyne chains up to IOC (straight or branched) independently containing either one or multiple ester (R is defined in paragraphs ii and iii above), carboxylic acids, ketone (R is defined in paragraphs i, ii and iii above), aldehyde, alcohols, amine
  • the ring structures above may have R groups (defined in parts i-vii and ix-xv) substituted at any position on the ring structure, have varying degrees of unsaturation, and be attached to any position on the steroid directly (for example, at a spiro ring junction or at a heteroatom) or tlirough an alkyl or hetero or alkyl hetero chain, and where chemically possible to one skilled in the art; ix) sulfate, sulfoxide, sulfamate, sulfone, sulfide, disulfide; x) phosphate, phosphonate; xi) nitro; xii) amides substituted with any R group defined in paragraphs i, ii and iii above, attached to the steroid through either the carbonyl carbon or amide nitrogen, or linked to the steroid by an R group as defined in paragraphs ii and iii above; xiii) any halogen containing al
  • Further evaluation of these compounds can include: in vitro evaluation for antitumor, antiproliferative or antiangiogenic activity using assays such as: in vitro tumor cell line or endothelial cell proliferation assays analyzed by direct cell counts, commercial kits measuring cellular metabolic function including MTT and XTT, or cell counts using metabolic incorporation into DNA of labeled ( 3 H-thymidine) or immunoreactive nucleotide (BrdU); in vitro assay of motility or migration including trans-membrane migration or endothelial cell layer wounding; surrogate in vitro assays for specific functions of 2ME2 analogs such as tubulin polymerization or SOD or other enzyme binding or inhibition assays; in vitro assays for induction of apoptosis or other perturbation of cell function including TUNEL and histone analysis, oxygen radical levels, p53 levels or p53 phosphorylation, or analysis of levels or activation state of enzymes in the apoptotic pathway such as caspases
  • Examples of further analyses which can be used to determine the suitability of these analogs for use in particular diseases and pathologies include: estrogenic activity which can be assessed in vitro using estrogen dependant MCF-7 proliferation assay, or in animal assays such as uterine weight gain or uterine or vaginal cytology or diestrus time perturbation; metabolic stability which can be analyzed using liver microsomes in vitro, or dosing animals or human subjects and measuring metabolism of the compound or formation of specific metabolites such as oxidation or demethylation products or conjugates using analytical techniques including HPLC, LCMS, GCMS, or LCMSMS; models of inflammation-associated angiogenesis including psoriasis, granuloma and collagen-induced arthritis models; the ApoE -/- knockout mouse model of atherosclerotic angiogenesis; porcine model of restenosis injury; neonatal mouse model of hypoxia-driven retinopathy; measurement of cholesterol levels; assays for antiangiogenic effects on fertility or reproduction or endometriosis
  • one embodiment of the invention includes the modifications listed above at the 17 position and also modifies the methyl ether of 2-methoxyestradiol so that it can not be a substrate for demethylases. Additionally, it has been claimed (U.S. Patent No. 5,504,074) and demonstrated (Cushman et al J. Med. Chem. 1995, 38, 2041-2049) that other electron-rich groups at the 2-position of estradiol (propyne, propene, ethoxy) have good anti-proliferative activity in vitro.
  • estradiol such as formyl, acetyl, methanol, 1-ethanol, 2-ethanol, amino, alkylamino, dialkyl amino, methyleneamine, methylene alkyl amine and methylene dialkylamine, and alkyl amide are anti-proliferative and antiangiogenic agents which have reduced or removed estrogenic activity and cannot be metabolized to 2-HO-E by demethylases.
  • Alkyl is defined as any carbon chain up to 10 carbons in length that is branched or straight. Listed below in Table 2 are data of 2-modified estradiol derivatives in HUNEC, MDA-MB-231 and MCF7 proliferation data.
  • one embodiment of the invention includes the modifications listed above at the 17 position and also modifies the 16 position of 2-methoxyestradiol. Examples of analogs modified at the 16 position are shown in Table 3.
  • formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example, those suitable for oral administration may include flavoring agents.
  • R and R 0 are independently -H, -Cl, -Br, -I, -F, -CN, lower alkyl, -OH, -OR6,-
  • R6 and R7 are independently hydrogen or an alkyl or branched alkyl with up to 10 carbons;
  • Z' is >CH, >COH, CR 2 , >C-R2-OH, >C-C ⁇ N, >CRl, where R2 is H or a straight or branched alkyl with up to 10 carbons or aralkyl, and in any position may have a hetero substitution in or on the carbon chain by hetero group as defined earlier, or where R2 is an alkyl or branched alkyl with up to 10 carbons, or aralkyl (also referred to herein as arylalkyl), or a hetero group wherein the hetero group may have more than one hetero atom and may be substituted, and Ri is -OH, -NH2, -Cl, -Br, -I, -F or -CF3 when Ri is terminal;
  • Rg is Rgi and Rg 2 , and wherein Rgi may be present or absent and when present is -H, an alkyl, alkenyl, or alkynyl of 1-10 carbon atoms that is straight chain or branched and is optionally substituted, and Rg 2 is a hetero group, wherein when Rgi is absent the heterogroup is bonded to the 17-position with a double bond, and wherein either Rgi or Rg 2 can be in the ⁇ position with the other group in the ⁇ position, and Ri is -OH, -NH2, -Cl, -Br, -I, -F or CF3 when Ri is terminal;
  • lower alkyl is defined as a carbon chain having 1-10 carbon atoms which may be branched or unbranched and wherein chemically possible to one skilled in the art.
  • preferably Z" is >COH or >C-OAc. In some embodiments of the invention, preferably Z" is >CH2- In some embodiments of the invention, preferably Rb and Ro are H.
  • terminal is defined as “at the end of a chain”.
  • the compounds of the present invention may also be presented as a pharmaceutically acceptable salts.
  • heterogroups that may be used in Rg 2 include, but are not limited to, ether groups, amino groups, carbonyl groups, haloalkyl, dihaloalkyl, or trihaloalkyl groups, hydroxy groups, ester groups, dialkylamino, or monoalkylamino groups, thiol, thioether, or thioester
  • Scheme 20 describes the coupling of 17-methylenehydroxy and 17-carboxyacids to 2- methoxyestradiol.
  • 2-methoxy-17(20)-methyleneestra-l,3,5(10)-triene-3-ol was protected as a methoxymethyl ether.
  • Hydroboration generally conditions: Mayo et al Microscale Organic Laboratory , 1986, ppl32, John Wiley & Sons, NY, NY.
  • 17-methylenehydroxy estratriene derivative deprotection gives 2- methoxy-17-methylenehydroxyestra-l,3,5(10)-triene-3-ol.
  • EXAMPLE 7 Synthesis of Esfradiol (E 2 ) Derivatives and modifications at the 2 position Synthesis of the E2 derivatives described herein is within the capability of one ordinarily skilled in the art. A specific description of the synthesis of the E2 derivatives having modifications at the 2 position and analogs discussed herein can be found in M. Cushman, H-M. He, J.A. Katzenellenbogen, CM. Lin and E. Hamel, Synthesis, antitubulin and antimitotic activity, and cytotoxicity of 2-methoxyesfradiol, and endogenous mammalian metabolite of esfradiol that inhibits tubulin polymerization by binding to the colchicine binding site, J. Med.
  • Oxalyl chloride (38 mmol, 19 mL, 2M, methylene chloride) was added to anhydrous methylene chloride (25 mL) and cooled to -46°C Methyl sulfoxide (5.40 mL, 76 mmol) was added dropwise, and the mixture was stirred for 2 minutes.
  • 3-Benzyl-2-methoxyestradiol in methylene chloride/methyl sulfoxide (10 mL/15 mL) and added within 5 minutes and the resulting mixture was stirred for 1 h.
  • Triethyl amine (170 mmol, 23.5 mL) was added drop- wise, stirred 5 minutes and warmed to rt.
  • Lithium diisopropyl amide (2M, Aldrich, heptane/THF/ethylbenzene) was dissolved in THF and cooled to -78°C, and 3-benzyl-2-methoxyestrone in THF (10 mL) was added dropwise. Following addition, the mixture was warmed to 0°C and stirred 1 hour (h). The mixture was then cooled to -78°C and DMPU (lmL) followed by crotyl bromide (205 ⁇ L, 2.0 mmol) were added dropwise. The mixture was warmed to rt over 4 h.
  • 3-benzyl-2-methoxyesfrone (1.51 g, 3.87 mmol) was suspended in tert-butoxy bis(dimethylamino)methane (1.64 mL, 8.13 mmol) and heated in an oil bath (155°C) for 1.5 h, during which time the steroid dissolved.
  • the reaction mixture was cooled to rt, and poured into ice water (100 mL) and washed with methylene chloride (2 x 100 mL). The organics were washed with brine (100 mL) dried with magnesium sulfate, filtered and rotoevaped to give product which was used without further purification (1.82 g, quanitative yield).
  • 16 ⁇ -crotyl-3-benzyl-2-methoxyesfrone (680 mg, 1.53 mmol) was dissolved in anhydrous THF (10 mL), and cooled to -78°C Lithium aluminum hydride (3.06 mmol, 116 mg) was added and the. solution was stirred for 2 h. The reaction was quenched by carefully adding water (2 mL) and warming to rt, then adding additional 50 mL portion of water. The mixture was washed with ethyl acetate (2 x 50 mL) and the combined organics were washed with water (50 mL), brine (50 mL), dried with magnesium sulfate, filtered and rotoevaped. The mixture was purified with 3:1 hexane:ethyl acetate SiO 2 Biotage FLASH apparatus to give 500 mg purified product (1.12 mmol, 73% yield).
  • EXAMPLE 18 Representative debenzylation of 16-alkyl-13-benzyl-2-methoxyesfradiol (Scheme 11) 16 ⁇ -crotyl-3-benzyl-2-methoxyesfradiol (500 mg, 1.12 mmol) was dissolved in ethyl acetate (25 mL) in Parr reaction bottle. The bottle was flushed with argon, and Pd/C (10%, 2.5 g) was added. The bottle was fitted to a Parr hydrogenator, filled and purged with hydrogen five times, pressurized to 50 psi, and agitated for 24 h. The mixture was filtered through a celite pad, rotoevaped and purified with a 3:1 hexane ethyl acetate SiO 2 flash column. Obtain 358 mg product (1.0 mmol, 89%).
  • Table 3 shows the results of antiproliferative activity tests of 2-methoxyesfradiol analogs of the present invention modified at the 16 position in cells and tumor.
  • MDA-MB-231 human breast carcinoma cells were grown in DMEM containing 10% FCS (Hyclone Laboratories, Logan UT) and supplemented with 2 mM L-Glutamine, 100 units/ml penicillin, 100 ⁇ g/ml streptomycin (Irvine Scientific, Santa Anna, CA).
  • MDA-MB-231 cells were plated at 5000 cells/ml in 96-well plates. After allowing the cells to attach overnight, the appropriate fresh media were applied containing differing concentrations of 2-ME2 or derivatives thereof, as described below. Drug was dissolved in DMSO (Sigma, St. Louis, MO) and added to the wells in a volume of 200 ⁇ l. Cells were incubated for two days at 37°C; at 32 h BrdU was added. BrdU cell proliferation assay (a nucleotide analogue that is incorporated into D ⁇ A) was performed as described by the manufacturer (Roche). Each condition was prepared in triplicate and the experiments were carried out a minimum of two times. Results presented are means +/- SE.
  • HUNEC cells were grown in EGM (Clonetics). Proliferation Assays HUVEC cells were plated at 5000 cells/ml in 96-well plates. After allowing the cells to attach overnight, the cells were washed with PBS and incubated in the absence of growth factor for 24 h (EBM, 2% FCS, Clonetics). Cells were treated with increasing concentrations of drug in EBM containing 2% FCS and lOng/ml bFGF for 48 h at 37°C. Drug preparation, volumes added and BrdU proliferation assay were performed as indicated above. 2-Methoxyestradiol is a potent anti-angiogenic and anti-tumor agent.
  • the MDA-MB-231 tumor cell line has a much greater sensitivity to substitutions at position 16 compared to HUNEC cells. Any group at position 16 larger than ethyl has a significant decrease in antiproliferative activity (IC50 >22 ⁇ M). 16 ⁇ -methyl has better activity than 2-methoxyestradiol, whereas 16 ⁇ -methyl (which had a 1:2 mixture of ⁇ : ⁇ ) has about 5-fold less activity than 2-methoxyesfradiol, and racemic 16-ethyl has about a 3-fold drop in activity compared to 2-methoxyestradiol.
  • MCF7 cells an estrogen dependent breast carcinoma cell line, were maintained in DMEM F12 (1:1) containing 10% (v/v) fetal bovine serum (Hyclone Laboratories, Logan, UT) and IX antibiotic-antimytotic. MCF7 cells were used between passage 60 and passage 90.
  • MCF7 estrogen-dependent proliferation assay the cells were seeded in complete media at 20-30,000 cells/well in a 24 well plate. After allowing the cells to adhere overnight the seeding density was determined by cell counts. Cells were washed with PBS (37°C) and starved by placing them in IMEM-phenol red free media containing 2% charcoal-dextran fetal bovine- stripped serum (Georgetown University) and IX antibiotic-antimitotic.
  • 16 ⁇ -propyl is more than ten-fold less active in inhibiting tumor growth while it has good activity inhibiting endothelial cell proliferation.
  • Other examples include: 16 ⁇ -propyl (4-fold difference), 16 ⁇ -i-butyl (5-fold difference), 16 ⁇ -n-butyl (4-fold difference) and 16 ⁇ -methanol (10-fold difference).
  • a small alkyl group at position 16 can be added without significantly altering the anti-proliferative activity of the molecule.
  • reaction scale was doubled and ethyl triphenylphosphonium bromide was used, from 2-methoxyestrone (613 mg, 2.04 mmol) obtain 540 mg (1.73 mmol, 84 % yield) of final product.
  • reaction scale was doubled and propyl triphenylphosphonium bromide was used, from 2-methoxyestrone (614.2 mg, 2.04 mmol) obtain 358.9 mg (1.10 mmol, 54% yield) of final product.
  • reaction scale was doubled and butyl triphenylphosphonium bromide was used, from 2-methoxyestrone (593.6 mg, 1.97 mmol) obtain 532.1 mg (1.56 mmol, 79% yield) of final product.
  • 17-Methylene analog (471.9 mg, 1.58 mmol) was dissolved ethyl acetate (20 ml). Pd/C 10% (47.5 mg) was added and reaction mixture was then subjected to hydrogenation in Parr hydrogenater for an hour under 30 psi of hydrogen. Reaction mixture was then filtered through Celite and solvent was removed via rotary evaporation to yield 472.5 mg white crystals (1.57 mmol, 99% yield) of the final product 2-methoxy-17 ⁇ -methylesfra-l,3,5(10)- triene-3-ol.
  • EXAMPLE 48 Synthesis of esfra-l,3,5(10)-triene-3-ol Into a stirring suspension of estrone (8.1 g, 30 mmols) in 60mL diethylene glycol, 20 mL 1-butanol and 2 mL hydrazine anhydrous (60mmols) were added. The reaction mixture was heated under reflux for 1 hour to get clear solution. After cooling reaction mixture to 50 °C, 5.04 g KOH pellets (90mmols) were added and butanol was distilled. The reaction mixture was heated at 50 °C for 2 hours and then cooled to RT.
  • EXAMPLE 54 Synthesis of 3-Azidoesfra-l,3,5(10)-triene-3-ol Into a solution ⁇ f 2-amino-esfradiol (144 mg, 0.5 mmols) in 3 mL acetic acid glacial, a solution of sodium nifrite (48 mg, 0J mmols) in 1 mL water was added at 0 °C. The color of the reaction mixture changed to orange-yellow. After stirring at 0 °C for 30 min. a solution of sodium azide (45 mg, 0.7 mmols) in water was added. The color of the reaction mixture changed to orange-red. Temperature was maintained at 0 °C for 30 min.
  • references for examples 31-54 include: Org. Synt. Coll. Vol. 5, 552; Org. Synt. Coll. Vol. 3, 590; and Shah, et. al. J. Med. Chem. 1995, 38, 4284.

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WO2002042319A3 (en) 2003-03-13
CA2430100A1 (en) 2002-05-30
WO2002042319A9 (en) 2005-03-31
AU8838601A (en) 2002-06-03
WO2002042319A2 (en) 2002-05-30
AU2001288386B2 (en) 2006-11-02

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