WO2002042319A9 - 2-substituted estrogens as antiangiogenic agents - Google Patents
2-substituted estrogens as antiangiogenic agentsInfo
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- WO2002042319A9 WO2002042319A9 PCT/US2001/026490 US0126490W WO0242319A9 WO 2002042319 A9 WO2002042319 A9 WO 2002042319A9 US 0126490 W US0126490 W US 0126490W WO 0242319 A9 WO0242319 A9 WO 0242319A9
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- 0 *N1CCCCC1 Chemical compound *N1CCCCC1 0.000 description 10
- DNXHEGUUPJUMQT-GUZDXLFXSA-N C[C@](CC1)(C(CC2)C(CCc3c4)C1c3ccc4O)C2=O Chemical compound C[C@](CC1)(C(CC2)C(CCc3c4)C1c3ccc4O)C2=O DNXHEGUUPJUMQT-GUZDXLFXSA-N 0.000 description 2
- OEQVLNZZOQCWAQ-IDQKJFIUSA-N C[C@](CC1)(C(CC2)C(CCc3c4)C1c3cc(OC)c4O)C2=C Chemical compound C[C@](CC1)(C(CC2)C(CCc3c4)C1c3cc(OC)c4O)C2=C OEQVLNZZOQCWAQ-IDQKJFIUSA-N 0.000 description 1
- NSRWXLSLADSLRV-JYNXCRBUSA-N C[C@](CC1)(C(CC2)C(CCc3c4)C1c3cc(OC)c4OCc1ccccc1)/C2=N/N(C)C Chemical compound C[C@](CC1)(C(CC2)C(CCc3c4)C1c3cc(OC)c4OCc1ccccc1)/C2=N/N(C)C NSRWXLSLADSLRV-JYNXCRBUSA-N 0.000 description 1
- BIOBFFLKMVXKGI-JNCCZOJWSA-N C[C@](CC1)(C(CC2)C(CCc3c4)C1c3cc(OC)c4OCc1ccccc1)C2=O Chemical compound C[C@](CC1)(C(CC2)C(CCc3c4)C1c3cc(OC)c4OCc1ccccc1)C2=O BIOBFFLKMVXKGI-JNCCZOJWSA-N 0.000 description 1
- FQYTXBHPRWKZOY-MLEUJMSOSA-N C[C@](CCC1)(CC2)C1C(CCc1c3)C2c1cc([N+]([O-])=O)c3O Chemical compound C[C@](CCC1)(CC2)C1C(CCc1c3)C2c1cc([N+]([O-])=O)c3O FQYTXBHPRWKZOY-MLEUJMSOSA-N 0.000 description 1
- HJKVPZJVBHWFCQ-HLYMMOCJSA-N C[C@](CCC1)(CC2)C1C(CCc1c3)C2c1ccc3O Chemical compound C[C@](CCC1)(CC2)C1C(CCc1c3)C2c1ccc3O HJKVPZJVBHWFCQ-HLYMMOCJSA-N 0.000 description 1
- YEJRWHAVMIAJKC-UHFFFAOYSA-N O=C1OCCC1 Chemical compound O=C1OCCC1 YEJRWHAVMIAJKC-UHFFFAOYSA-N 0.000 description 1
- OZJPLYNZGCXSJM-UHFFFAOYSA-N O=C1OCCCC1 Chemical compound O=C1OCCCC1 OZJPLYNZGCXSJM-UHFFFAOYSA-N 0.000 description 1
Classifications
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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
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- 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
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- 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
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- 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]
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- 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
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- 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
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- 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-methoxyesfradiol (2ME 2 ) and their effect on diseases characterized by abnormal cell mitosis and/or abnormal angiogenesis.
- 2ME 2 2-methoxyesfradiol
- 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 form 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.
- a disease mediated by angiogenesis is 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.
- 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, neovascular glaucoma, and refrolental fibroplasia.
- Other diseases associated with corneal neovascularization include, but are not limited to, epidemic keratoconjunctivitis, Vitamin 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, chrome 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, Best's disease, myopia, optic pits, Stargardt's disease, pars planitis, chronic retinal detachment, hyperviscosity syndromes, toxoplasmosis, trauma and post-laser complications.
- 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 vifreoretinopathy.
- Another 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 confributes 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.
- 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. The hypothesis that tumor growth is angiogenesis-dependent was first proposed in 1971.
- 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.
- Examples of the indirect evidence which support this concept include: (1) The growth rate of tumors implanted in subcutaneous transparent chambers in mice is slow and linear before neovascularization, and rapid and nearly exponential after neovascularization. (Algire, et al., J. Nat. Cancer Inst., 6:73-85 (1945)). (2) Tumors grown in isolated perfused organs where blood vessels do not proliferate are limited to 1-2 mm 3 but expand rapidly to >1000 times this volume when they are transplanted to mice and become neovascularized. (Folkman, et al, Annals of Surgery,
- 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 . 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).
- tumors are implanted on the chick embryo chorioallantoic membrane, they grow slowly during an avascular phase of >72 hours, but do not exceed a mean diameter of
- 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
- a specific antibody against VEGF reduces microvessel density and causes "significant or dramatic" inhibition of growth of three human tumors which rely on VEGF as their sole mediator of angiogenesis (in nude mice). The antibody does not inhibit growth of the tumor cells in vitro.
- 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)). (10) Infraperitoneal 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.
- 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 microvascular 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.
- 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 tumors progress to large cavernous and infilfrative forms and create clinical complications. Systemic fo ⁇ ns 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.
- angiogenesis is an important step in ovulation and also in implantation of the blastula after fertilization.
- angiogenesis could be used to induce amenorrhea, to block ovulation, or to prevent implantation by the blastula.
- excessive repair or fibroplasia can be a detrimental side effect of surgical procedures and may be caused or exacerbated by angiogenesis.
- Adhesions are a frequent complication of surgery and lead to problems such as small bowel obstruction.
- Taylor, et al. (Nature, 297:307 (1982)) have used protamine to inhibit angiogenesis. The toxicity of protamine limits its practical use as a therapeutic. Folkman, et al. (Science, 221:719 (1983), and U.S. Pat. Nos.
- Interferon beta is also a potent inhibitor of angiogenesis induced by allogeneic spleen cells.
- Human recombinant interferon (alpha/A) was reported to be successfully used in the freatment of pulmonary hemangiomatosis, an angiogenesis-induced disease.
- Other agents which have been used to inhibit angiogenesis include ascorbic acid ethers and related compounds. (Japanese Kokai Tokkyo Koho No.58-13 (1978)). Sulfated polysaccharide DS 4152 also inhibits angiogenesis.
- Additional anti-angiogenic compounds include Angiostatin® (U.S. Patent Nos. 5,639,725; 5,792,845; 5,885,795; 5,733,876; 5,776,704; 5,837,682; 5,861,372, and 5,854,221) and EndostatinTM (U.S. Patent No. 5,854,205).
- Angiostatin® U.S. Patent Nos. 5,639,725; 5,792,845; 5,885,795; 5,733,876; 5,776,704; 5,837,682; 5,861,372, and 5,854,221
- EndostatinTM U.S. Patent No. 5,854,205
- Another compound which has been shown to inhibit angiogenesis is thalidomide. (D'Amato, et al, Proc. Natl. Acad. Sci., 90:4082-85 (1994)).
- Thalidomide is a hypnosedative that has been successfully used to freat 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. Dermatol, 127 Suppl, 40:67-8 (1992); Gunzler, Med.
- 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.
- 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.
- thalidomide 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-Methoxyesfradiol is an endogenous metabolite of esfradiol (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.
- the present invention relates to analogs of 2-methoxyesfradiol 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, abnonnal 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., athe
- compositions described above can be used to block ovulation and implantation of a blastula or to block menstruation (induce amenorrhea).
- 2-methoxyestradiol (2ME 2 )
- an endogenous metabolite of esfradiol with no intrinsic esfrogenic 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.
- 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. Replacement of 2-methoxy group by other moieties such as 2-N-formamide or 2- fo ⁇ nyl and 2-N.N-dimethylamino retained antiproliferative activity, but these groups cannot be de-methylated to yield the esfrogenic 2-hydroxyl derivatives. These analogs have selective in vitro antiproliferative activity for endothelial cells over the tumor cell line assessed.
- compounds that are useful in accordance with the invention include novel 2-methoxyesfradiol derivatives that exhibit anti-mitotic, anti-angiogemc and anti-tumor properties. Specific compounds according to the invention are described below. Preferred compounds of the invention are 2-methoxyesfradiol derivatives modified at the 2, 16, or 17 positions or combinations thereof. Those skilled in the art will appreciate that 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-Methoxyesfradiol is an endogenous metabolite of esfradiol 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-Methoxyesfradiol is metabolized to a less active metabolite, 2-methoxyesfrone (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 esfradiol.
- the enzymes responsible for this reaction on esfradiol 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
- Each of the four members of this enzyme family, types 1, 2, 3, and 4 have distinct activity.
- 17 ⁇ - HSD type 1 catalyzes the reductive reaction (estrone to esfradiol)
- 17 ⁇ -HSD type 2 catalyzes the oxidation reaction (esfradiol to estrone)
- type 3 catalyzes 4-androstenedione to testosterone.
- an additional metabolic deactivation pathway results in conjugation of 2-methoxyesfradiol or 2-methoxyesfrone with molecules such as sulfate or glucuronic acid and subsequent loss via excretion.
- 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 ⁇ -hydroxysteroid 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-methoxyesfradiol may also retard or prevent conjugation, such as glucuronidation.
- 2-Methoxy-17-deoxyesfrone, 17-ethyl-2-methoxyesfrone and 17-methyl-2- methoxyestrone analogs showed equal or better antiproliferative activity than 2ME 2 but have diminished the potential to change either into 2-methoxyesfrone or to conjugate at position-17.
- Increasing carbon chain length at-position-17 of 2-methoxyestrone decreases the antiproliferative activity.
- Some position-2 modified 17-deoxyesfrone analogs retained good antiproliferative activity in HUVEC cells only, suggesting that these analogs may be potent anti-angiogenics in vivo.
- Anti-Proliferative activity is evaluated in situ by testing the ability of an improved esfradiol 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.
- a 100 ⁇ g disk of the esfradiol 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.
- 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.
- a reaction mixture typically contains 1.0M monosodium ' glutamate (pH 6.6), 1.0 mg/ml (10 ⁇ M) tubulin, 1.0 mM MgCl2, 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 specfrophotometer is set at 37°C Microtubule assembly is evaluated by increased turbity at 350 nm. Alternatively, inhibition of microtubule assembly can be followed by fransmission 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 freat 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 (Osler-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, neovascular glaucoma and retrolental ibroplasias, epidemic keratoco ⁇ junctivitis, 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 freated 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 detacliment, hyperviscosity syndromes, toxoplasmosis, frauma 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 freat 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 esfrogenic and non-esfrogenic biochemical pathways.
- Prodrug also relates to conjugated prodrugs and uses thereof. More particularly, the invention relates to conjugates of esfradiol compounds such as
- the present invention provides a conjugated prodrug of an esfradiol compound, preferably of 2-methoxyesfradiol or a functionally active analogue or derivative thereof, conjugated to a biological activity modifying agent.
- the analogue or derivative of 2-methoxyestradiol has one or more of the biological activities of 2-methoxyesfradiol.
- 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.
- the conjugated prodrug according to the present invention includes 2-methoxyestradiol or a functionally active analogue or derivative thereof, conjugated to a peptide moiety.
- an esfradiol 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 freatment 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 hyperfrophic growth or inflammation including administering to a patient in need of such prophylaxis or freatment 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 freated, 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.
- 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 admimstered 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.
- the 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, mfrafracheal, 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).
- 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.
- 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
- 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.
- 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.
- 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 esfradiol (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. In vitro data suggests that 2-methoxyestradiol does not engage the estrogen receptor for its anti-proliferative activity. However, the presence of demethylases in vivo may metabolize this compound to 2-hydroxyesfradiol, which has been shown to be esfrogenic by several approaches.
- the present invention improves the bioavailability of esfradiol or 2- methoxyestradiol and reduces the formation of esfrogenic 2-methoxyestradiol metabolities.
- the present invention modifies esfradiol 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.
- alkyls both straight and branched up to ten carbons, having either the alpha or beta stereochemistry, and may be saturated or unsaturated, substituted or unsubstituted
- alkynyls with either straight or branched alkyl chains, up to ten carbons; and may be saturated or unsaturated, substituted or unsubstituted, with the C ⁇ C at any position
- d) wherein aromatic or hetero groups can be
- 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. 100% pure isomers are contemplated by this invention, however a stereochemical isomer labeled as ⁇ or ⁇ may be a mixture of both in any ratio, where it is chemically possible by one skilled in the art.
- 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, alken
- 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 10C (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 through 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 alkyl, 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: esfrogenic activity which can be assessed in vitro using esfrogen 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
- one embodiment of the invention includes the modifications listed above at the 17 position and also modifies the methyl ether of 2-methoxyesfradiol 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 esfradiol (propyne, propene, ethoxy) have good anti-proliferative activity in vitro.
- modifications at C-2 of esfradiol 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 esfrogenic activity and cannot be metabolized to 2-HO-E 2 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 esfradiol derivatives in HUVEC, MDA-MB-231 and MCF7 proliferation data.
- molecular modeling suggests that there may be a hydrogen bond that forms between the 3-hydroxy group and the methoxy group of 2- methoxyesfradiol. This interaction may be important for both 2-methoxyestradiol's anti- proliferative and anti-angiogenic action as well as its non-esfrogenic activity. It is disclosed that any group that can be placed at position 2 of esfradiol and has the potential to form a hydrogen bond with the 3-hydroxy group is an anti-proliferative and anti-angiogenic agent that lacks esfrogenic activity.
- 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.
- Rb and R 0 are independently -H, -Cl, -Br, -I, -F, -CN, lower alkyl, -OH, -OR6,-
- 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 R 0 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
- 2-alkynyl substituted analogs To prepare the 2-alkynyl substituted analogs, the propynyl group was introduced and the 3 -alcohol was protected as the tBDMS ether using Castro's conditions (Castro et al J Org. Chem. 1966, 31, 4071). Subsequent deprotection using TBAF gave 2-propynyl-17-alkyl-esfratriene analogs.
- 2-Alkene substituted analogs can be prepared by protecting the 3-alcohol as a methoxymethyl ether, subsequent 2-formylation (Lovely et al Tetrahedron Lett. 1994, 8735; Pert et al Aust J. Chem.
- 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.
- Methyl sulfoxide (5.40 mL, 76 mmol) was added dropwise, and the mixture was stirred for 2 minutes.
- Triethyl amine (170 mmol, 23.5 mL) was added drop- wise, stirred 5 minutes and warmed to rt. Water (-200 mL) was added and the mixture was washed with methylene chloride (3x 200 mL).
- EXAMPLE 11 Representative preparation of 16 ⁇ -alkyl-3-benzyl-2-methoxyestrone (Scheme 13) 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.
- the reaction was quenched by carefully adding water (100 mL) and washing with ethyl acetate (2 x 100 mL). The combined organics were washed with water (100 mL) and brine (100 mL). The solution was dried with magnesium sulfate, filtered and rotoevaped.
- the crude product was purified using hexane / ethyl acetate (9:1) SiO Biotage FLASH apparatus. 680 mg (1.53 mmol) of product was obtained and approximately 121 mg (0.31 mmol) of starting material was recovered (90% yield based on recovered starting material). Diastereomeric ratio of 16 ⁇ / ⁇ is approximately 2: 1 (s HI 8 signals at 0.88, 0.79 ppm).
- EXAMPLE 12 Representative preparation of 16-alkyl-16-methoxycarbonyl-3-benzyl-2-methoxyesfrone (Scheme 12) 3-benzyl-16-carbomethoxy-2-methoxyestrone (0.840 g, 1.87 mmol, prepared as in Example 15), potassium hydride (1.5 g, 10.9 mmol, 30% mineral oil dispursion, washed in hexanes) and 18-crown-6 (120 mg, 0.4 mmol) was mixed in THF (40 mL) and refluxed for 1 h. The mixture was cooled to rt, and allyl bromide (537 ⁇ L, 6.2 mmol) was added and the mixture was refluxed for 18 h.
- EXAMPLE 13 Representative decarboxylation of 16-alkyl-16-methoxycarbonyl-3-benzyl-2-methoxyestrone (Scheme 12) 16-allyl-16-carbomethoxy-3-benzyl-2-methoxyestrone (697 mg, 1.42 mmol), lithium chloride (1.15 g, 27 mmol), water (485 ⁇ L, 27 mmol) were dissolved in DMF (63 mL) and refluxed for 20 h.
- EXAMPLE 16 Representative procedure for preparation of 16-alkyl-3-benzyl-2-methoxyesfra-17 ⁇ -diol (Scheme 11) 16 ⁇ -crotyl-3-benzyl-2-methoxyestrone (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-methoxyestradiol (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.
- 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 concenfrations 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 inco ⁇ orated into DNA) 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.
- HUVEC 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-Methoxyesfradiol 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 HUVEC 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-methoxyesfradiol, whereas 16 ⁇ -methyl (which had a 1:2 mixture of ⁇ : ⁇ ) has about 5 -fold less activity than 2-methoxyestradiol, 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. For 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.
- EXAMPLE 33 Preparation of 2-methoxy- 17(20)-Z-propylideneesfra- 1 ,3 ,5 (10)-triene-3 -ol Reaction conditions as above except reaction scale was doubled and propyl triphenylphosphonium bromide was used, from 2-methoxyesfrone (614.2 mg, 2.04 mmol) obtain 358.9 mg (1.10 mmol, 54% yield) of final product.
- EXAMPLE 35 Representative procedure for preparation of 17-alkyl-2-methoxyestradiol analogs 2-methoxy-17 ⁇ -methylestra-l,3,5(10)-triene-3-ol (Table 1, entry 8) 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.
- EXAMPLE 48 Synthesis of estra-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-Azidoestra-l,3,5(10)-triene-3-ol Into a solution of 2-amino-esfradiol (144 mg, 0.5 mmols) in 3 mL acetic acid glacial, a solution of sodium nitrite (48 mg, 0.7 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. and then raised to RT.
- 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. All of the publications mentioned herein are hereby incorporated by reference in their entireties. The above examples are merely demonstrative of the present invention, and are not intended to limit the scope of the appended claims.
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CA002430100A CA2430100A1 (en) | 2000-11-27 | 2001-08-24 | Antiangiogenic agents |
AU8838601A AU8838601A (en) | 2000-11-27 | 2001-08-24 | Antiangiogenic agents |
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TWI320712B (en) * | 2001-09-05 | 2010-02-21 | Alcon Inc | The use of non-feminizing estrogens as retinoprotective agents for the treatment of glaucoma |
US20040214806A1 (en) | 2002-09-03 | 2004-10-28 | Iok-Hou Pang | Use of non-feminizing estrogens as retinoprotective agents for the treatment of glaucoma |
DE10307103A1 (en) | 2003-02-19 | 2004-09-09 | Schering Ag | Anti-tumor effective 2-substituted D-homostra-1,3,5 (10) -trien-3-yl sulfamate |
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US20050148496A1 (en) * | 2003-11-26 | 2005-07-07 | Entelos, Inc. | Treatment of rheumatoid arthritis with hypoxia inducible factor-1alpha antagonists |
TW200611909A (en) | 2004-08-04 | 2006-04-16 | Akzo Nobel Nv | Process for the preparation 2-substituted-derivatives of estrone and estradiol |
TW200613316A (en) | 2004-08-26 | 2006-05-01 | Akzo Nobel Nv | Process for the preparation of estrone and/or estradiol-derivates |
WO2014089177A2 (en) | 2012-12-04 | 2014-06-12 | Massachusetts Institute Of Technology | Compounds, conjugates and compositions of epipolythiodiketopiperazines and polythiodiketopiperazines |
US10201623B2 (en) | 2013-03-15 | 2019-02-12 | Memorial Sloan Kettering Cancer Center | HSP90-targeted cardiac imaging and therapy |
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US10918627B2 (en) | 2016-05-11 | 2021-02-16 | Massachusetts Institute Of Technology | Convergent and enantioselective total synthesis of Communesin analogs |
US11932650B2 (en) | 2017-05-11 | 2024-03-19 | Massachusetts Institute Of Technology | Potent agelastatin derivatives as modulators for cancer invasion and metastasis |
CN107746420B (en) * | 2017-09-28 | 2020-08-14 | 湖南新合新生物医药有限公司 | Preparation method of 16 beta alkyl steroid compound |
US10640508B2 (en) | 2017-10-13 | 2020-05-05 | Massachusetts Institute Of Technology | Diazene directed modular synthesis of compounds with quaternary carbon centers |
WO2020247054A1 (en) | 2019-06-05 | 2020-12-10 | Massachusetts Institute Of Technology | Compounds, conjugates, and compositions of epipolythiodiketopiperazines and polythiodiketopiperazines and uses thereof |
Family Cites Families (22)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3166577A (en) * | 1956-05-29 | 1965-01-19 | Syntex Corp | 1, 2-dimethyl estrogens and intermediates used in the production thereof |
GB857080A (en) * | 1956-05-29 | 1960-12-29 | Syntex Sa | Cyclopentanophenanthrene derivatives and process for the production thereof |
GB857081A (en) * | 1956-05-29 | 1960-12-29 | Syntex Sa | Cyclopentanophenanthrene derivatives and process for the production thereof |
US2846453A (en) * | 1957-05-28 | 1958-08-05 | Searle & Co | Alpha-ring-acylated estrone derivatives, and corresponding alcohols, their esters, and ethers |
US3562260A (en) * | 1965-08-23 | 1971-02-09 | Ormonoterapia Richter Spa | 2-carbonyl-estratrienes and method of their preparation |
DE2004516A1 (en) * | 1969-03-05 | 1970-09-10 | VEB Jenapharm, Jena | Thermpentic steroidal sulphonate esters |
US4917826A (en) * | 1985-10-18 | 1990-04-17 | The Upjohn Company | Cyclic hydrocarbons with an aminoalkyl sidechain |
JP2517561B2 (en) * | 1986-10-03 | 1996-07-24 | 新技術事業団 | Category Estrogen Immunoassay Kit |
JPH02502997A (en) * | 1987-04-16 | 1990-09-20 | ジ・アップジョン・カンパニー | Cyclic hydrocarbons with aminoalkyl side chains |
US5504074A (en) * | 1993-08-06 | 1996-04-02 | Children's Medical Center Corporation | Estrogenic compounds as anti-angiogenic agents |
EP0791660A1 (en) * | 1996-02-22 | 1997-08-27 | Smithkline Beecham Corporation | A novel diagnostic marker for splicing variants of genes associated with neurological function |
US5763432A (en) * | 1997-01-29 | 1998-06-09 | Sri International | Steriod inhibitors of estrone sulfatase and associated pharmaceutical compositions and methods of use |
US6136992A (en) * | 1997-03-13 | 2000-10-24 | The United States Of America As Represented By The Department Of Health And Human Services | 2-alkoxy estradiols and derivatives thereof |
CA2208916A1 (en) * | 1997-07-03 | 1999-01-03 | Hyal Pharmaceutical Corporation | Promotion of wound healing utilizing steroids having reduced deterioroussystemic side effects typical of glucocorticoids, mineralocorticoids andsex steroids |
US6054446A (en) * | 1997-12-24 | 2000-04-25 | Sri International | Anti-estrogenic steroids, and associated pharmaceutical compositions and methods of use |
US6046186A (en) * | 1997-12-24 | 2000-04-04 | Sri International | Estrone sulfamate inhibitors of estrone sulfatase, and associated pharmaceutical compositions and methods of use |
AR021757A1 (en) * | 1998-08-07 | 2002-08-07 | Endorech Inc | METHOD FOR INHIBITING THE CONVERSION OF 4-ANDROSTAN-3,17-DIONA IN TESTOSTERONE OR 3ALFA-ANDROSTAN-3,17-DIONA IN DIHYDROTESTOSTERONE, INHIBIT THE ACTIVITY OF 3ALFA-HYDROXYSTEROID DEHYDROGENASE OF TYPE 3 OF HIFTIVA OF TYPE 3 PUTATIVE INHIBITOR OF THE CONVERSION OF 4-ANDROSTEN-3 |
AU5687199A (en) * | 1998-08-24 | 2000-03-14 | Global Vascular Concepts, Inc. | Use of anti-angiogenic agents for inhibiting vessel wall injury |
IL145995A0 (en) * | 1999-04-30 | 2002-07-25 | Sterix Ltd | Use |
GB9910934D0 (en) * | 1999-05-11 | 1999-07-14 | Res Inst Medicine Chem | Chemical compounds |
AUPQ342599A0 (en) * | 1999-10-14 | 1999-11-04 | University Of Melbourne, The | Conjugates and uses thereof |
DK1287017T3 (en) * | 2000-05-11 | 2005-12-05 | Res Inst Medicine Chem | 2-substituted pregna-1,3,5 (10) -triene and chola-1,3,5 (10) triene derivatives and their biological activity |
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2001
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- 2001-08-24 WO PCT/US2001/026490 patent/WO2002042319A2/en active IP Right Grant
- 2001-08-24 JP JP2002544452A patent/JP2004537499A/en active Pending
- 2001-08-24 AU AU8838601A patent/AU8838601A/en active Pending
- 2001-08-24 AU AU2001288386A patent/AU2001288386B2/en not_active Ceased
- 2001-08-24 EP EP01968112A patent/EP1343803A2/en not_active Withdrawn
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WO2002042319A2 (en) | 2002-05-30 |
WO2002042319A3 (en) | 2003-03-13 |
JP2004537499A (en) | 2004-12-16 |
CA2430100A1 (en) | 2002-05-30 |
EP1343803A2 (en) | 2003-09-17 |
AU8838601A (en) | 2002-06-03 |
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