U.S. patent application number 11/519570 was filed with the patent office on 2007-01-11 for methods of treating disease states using antiangiogenic agents.
Invention is credited to Gregory E. Agoston, Theresa M. LaVallee, Victor S. Pribluda, Jamshed H. Shah, Anthony M. Treston.
Application Number | 20070010505 11/519570 |
Document ID | / |
Family ID | 34392892 |
Filed Date | 2007-01-11 |
United States Patent
Application |
20070010505 |
Kind Code |
A1 |
Agoston; Gregory E. ; et
al. |
January 11, 2007 |
Methods of treating disease states using antiangiogenic agents
Abstract
Compositions and methods for treating mammalian disease
characterized by undesirable angiogenesis by administering
compounds of the general formula: ##STR1## wherein the variables
are defined in the specification.
Inventors: |
Agoston; Gregory E.;
(Germantown, MD) ; LaVallee; Theresa M.;
(Rockville, MD) ; Pribluda; Victor S.; (Silver
Spring, MD) ; Shah; Jamshed H.; (Brookeville, MD)
; Treston; Anthony M.; (Rockville, MD) |
Correspondence
Address: |
JOHN S. PRATT, ESQ;KILPATRICK STOCKTON, LLP
1100 PEACHTREE STREET
ATLANTA
GA
30309
US
|
Family ID: |
34392892 |
Appl. No.: |
11/519570 |
Filed: |
September 12, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10856340 |
May 28, 2004 |
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11519570 |
Sep 12, 2006 |
|
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60474288 |
May 28, 2003 |
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Current U.S.
Class: |
514/182 ;
552/626 |
Current CPC
Class: |
A61P 13/02 20180101;
A61P 31/00 20180101; A61P 1/00 20180101; A61P 37/08 20180101; A61P
7/02 20180101; A61P 13/08 20180101; A61P 1/04 20180101; A61P 25/00
20180101; C07J 41/00 20130101; A61P 9/10 20180101; A61P 37/06
20180101; A61P 7/06 20180101; A61P 37/02 20180101; A61P 35/00
20180101; A61P 1/16 20180101; A61P 9/00 20180101; A61P 27/02
20180101; A61P 35/02 20180101; A61P 11/00 20180101; A61P 31/22
20180101; A61P 29/00 20180101; A61P 21/00 20180101; A61P 27/06
20180101; A61P 31/18 20180101; A61P 15/00 20180101; A61P 13/12
20180101; A61P 17/06 20180101; A61P 19/02 20180101; A61P 17/00
20180101; A61P 31/04 20180101; A61P 31/10 20180101 |
Class at
Publication: |
514/182 ;
552/626 |
International
Class: |
A61K 31/56 20060101
A61K031/56; C07J 1/00 20060101 C07J001/00 |
Claims
1. (canceled)
2. A method of inhibiting angiogenesis in a human or animal
comprising administering to the human or animal a compound having
the formula: ##STR241## wherein, R.sub.a is selected from
--OCH.sub.3; --OCH.sub.2CH.sub.3; or --CCCH.sub.3; Z' is selected
from >C--F, >C--NH.sub.2, >CCONH.sub.2, >C--NHCOH,
>C--OSO.sub.2NH.sub.2, or >C--CHCH.sub.2, and Z'' is selected
from >C(H.sub.2), >C(H)--CH.sub.3, >C.dbd.CH.sub.2,
>C.dbd.CHCH.sub.3, or >C.dbd.O; provided that when Z' is
>C--OSO.sub.2NH.sub.2 and Z'' is >C(H.sub.2) or >C.dbd.O,
R.sub.a is neither --OCH.sub.3 nor --OCH.sub.2CH.sub.3.
3. The method of claim 2, wherein the administration of the
compound is in a daily dose, a daily sub-dose, or any appropriate
fraction thereof to the human or animal.
4. The method of claim 2, wherein the amount of the compound
administered is approximately 0.1 to approximately 300
mg/kg/day.
5. The method of claim 2, wherein the amount of the compound
administered is approximately 0.5 to approximately 50
mg/kg/day.
6. The method of claim 2, wherein the amount of the compound
administered is approximately 1 to approximately 10 mg/kg/day.
7. The method of claim 2, wherein the administration of the
compound is oral, parenteral, transdermal, topical, intravenous,
subcutaneous, intramuscular, intradermal, ophthalmic, epidural,
intratracheal, sublingual, buccal, rectal, vaginal, nasal or
inhalation.
8. The method of claim 2, wherein the compound is administered in a
composition comprising an additive selected from an anti-oxidant, a
buffer, a bacteriostat, a liquid carrier, a solute, a suspending
agent, a thickening agent, a flavoring agent, a gelatin, glycerin,
a binder, a lubricant, an inert diluent, a preservative, a surface
active agent, a dispersing agent, a biodegradable polymer, or any
combination thereof.
9. The method of claim 2, wherein the compound is administered in
the form of a tablet, a capsule, a lozenge, a cachet, a solution, a
suspension, an emulsion, a powder, an aerosol, a suppository, a
spray, a pastille, an ointment, a cream, a paste, a foam, a gel, a
tampon, a pessary, a granule, a bolus, a mouthwash, or a
transdermal patch.
10. The method of claim 2, wherein the angiogenesis is associated
with diabetic retinopathy, retinopathy of prematurity, corneal
graft rejection, neovascular glaucoma, retrolental fibroplasias,
epidemic keratoconjunctivitis, Vitamin A deficiency, contact lens
overwear, atopic keratitis, superior limbic keratitis, pterygium
keratitis sicca, Sjogren's syndrome, 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, marginal
keratolysis, trauma, arthritis, rheumatoid arthritis,
polyarteritis, systemic lupus, Wegener's sarcoidosis, scleritis,
Stevens-Johnson disease, radial keratotomy, macular degeneration,
sickle cell anemia, sarcoid, pseudoxanthoma elasticum, Paget's
disease, vein occlusion, artery occlusion, carotid obstructive
disease, chronic uveitis, chronic vitritis, Lyme's disease, Eales'
disease, Behcet's disease, myopia, optic pits, Stargardt's disease,
pars planitis, chronic retinal detachment, hyperviscosity
syndromes, toxoplasmosis, post-laser complications, abnormal
proliferation of fibrovascular or fibrous tissue, hemangiomas,
Osler-Weber-Rendu disease, solid tumors, blood-borne tumors,
acquired immune deficiency syndrome, ocular neovascular disease,
age-related macular degeneration, osteoarthritis, diseases caused
by chronic inflammation, Crohn's disease, ulcerative colitis,
tumors of rhabdomyosarcoma, tumors of retinoblastoma, Ewing's
sarcoma, neuroblastoma, tumors of osteosarcoma, leukemia,
psoriasis, atherosclerosis, pemphigoid, infections causing
retinitis, infections causing choroiditis, presumed ocular
histoplasmosis, Best's disease, proliferative vitreoretinopathy,
Bartonellosis, acoustic neuromas, neurofibroma, trachoma, or
pyogenic granulomas.
11-95. (canceled)
96. A method of treating an eye condition in a human or an animal
comprising administering to the human or animal a compound having
the formula ##STR242## ##STR243## ##STR244## ##STR245## ##STR246##
##STR247## ##STR248## ##STR249## ##STR250## ##STR251## ##STR252##
##STR253##
97. The method of claim 96, wherein the eye condition is ocular
neovascular disease, diabetic retinopathy, retinopathy of
prematurity, macular degeneration, age related macular
degeneration, corneal graft rejection, neovascular glaucoma,
retrolental fibroplasias, epidemic keratoconjunctivitis, contact
lens overwear, atopic keratitis, superior limbic keratitis,
pterygium keratitis sicca, myopia, chronic retinal detachment,
optic pits, Terrien's marginal degeneration, hyperviscosity
syndromes, chronic uveitis, chronic vitritis, presumed ocular
histoplasmosis, retinitis, choroiditis, proliferative
vitreoretinopathy, scleritis, Eales' disease, Best's disease,
trachoma, pars planitis, due to chronic inflammation, due to
post-laser complications, or due to radial keratotomy.
98-124. (canceled)
125. A method of treating an inflammatory or immune mediated
disease in a human or an animal comprising administering to the
human or animal a compound selected from ##STR254## ##STR255##
##STR256## ##STR257## ##STR258## ##STR259## ##STR260## ##STR261##
##STR262## ##STR263## ##STR264## ##STR265##
126. The method of claim 125, wherein the inflammatory or immune
mediated disease is rheumatoid arthritis, osteoarthritis,
ulcerative colitis, Crohn's disease, Mooren's ulcer, arthritis,
sarcoidosis, inflammatory bowel diseases, immune mediated bowel
disease, systemic lupus, pars planitis, Wegener's syndrome,
Stevens-Johnson disease, Behcet's disease, pemphigoid, Lyme's
disease, or acquired immune deficiency syndrome.
127-138. (canceled)
139. A method of treating an infectious disease in a human or an
animal comprising administering to the human or animal a compound
selected from ##STR266## ##STR267## ##STR268## ##STR269##
##STR270## ##STR271## ##STR272## ##STR273## ##STR274## ##STR275##
##STR276## ##STR277##
140. The method of claim 139, wherein the infectious disease is
syphilis, a bacterial infection, a mycobacterial infection, a
bacterial ulcer, a fungal ulcer, a Herpes simplex infection, a
Herpes zoster infection, a protozoan infection, a Bartonellosis
infection, or toxoplasmosis.
141-149. (canceled)
150. A method of treating a cancerous disease in a human or an
animal comprising administering to the human or animal a compound
selected from ##STR278## ##STR279## ##STR280## ##STR281##
##STR282## ##STR283## ##STR284## ##STR285## ##STR286## ##STR287##
##STR288## ##STR289##
151. The method of claim 150, wherein the cancerous disease is
rhabdomyosarcoma, retinoblastoma, Ewing sarcoma, neuroblastoma,
acoustic neuroma, osteosarcoma, neurofibroma, hemangioma, breast
cancer, prostrate cancer, renal cell cancer, a brain tumor, ovarian
cancer, colon cancer, liver cancer, lung cancer, bladder cancer,
cutaneous melanoma, Kaposi's sarcoma, or leukemia.
152-166. (canceled)
167. A method of treating a blood or blood vessel disease in a
human or an animal comprising administering to the human or animal
a compound selected from ##STR290## ##STR291## ##STR292##
##STR293## ##STR294## ##STR295## ##STR296## ##STR297## ##STR298##
##STR299## ##STR300## ##STR301##
168. The method of claim 167, wherein the blood or blood vessel
disease is vein occlusion, artery occlusion, carotid obstructive
disease, polyarteritis, atherosclerosis, Osler-Weber-Rendu disease,
sickle cell anemia, leukemia, an acute neoplastic disease of the
bone marrow, a chronic neoplastic disease of the bone marrow, a
hemangioma, a hereditary hemorrhagic telangiectasia, a disease of
the bone marrow, anemia, impaired blood clotting, enlargement of
the lymph nodes, enlargement of the liver, or enlargement of the
spleen.
169-175. (canceled)
176. The method of claim 168, wherein the acute or chronic
neoplastic disease of the bone marrow is multiple myeloma.
177. The method of claim 168, wherein the acute or chronic
neoplastic disease of the bone marrow is myelo dysplastic
syndrome.
178-183. (canceled)
184. A method of treating a skin condition in a human or an animal
comprising administering to the human or animal a compound selected
from ##STR302## ##STR303## ##STR304## ##STR305## ##STR306##
##STR307## ##STR308## ##STR309## ##STR310## ##STR311## ##STR312##
##STR313##
185. The method of claim 184, wherein the skin condition is
abnormal wound healing, acne rosacea, due to chemical burns, or
psoriasis.
186-188. (canceled)
189. A method of treating a tumor in a human or an animal
comprising administering to the human or animal a compound having
the formula ##STR314## ##STR315## ##STR316## ##STR317## ##STR318##
##STR319## ##STR320## ##STR321## ##STR322## ##STR323## ##STR324##
##STR325##
190. The method of claim 189, wherein the tumor is a blood borne
tumor, a cancerous blood borne tumor, a solid tumor, a benign
tumor, or a cancerous tumor.
191-194. (canceled)
195. A method of inhibiting abnormal cell mitosis in a human or
animal comprising administering to the human or animal a compound
selected from ##STR326## ##STR327## ##STR328## ##STR329##
##STR330## ##STR331## ##STR332## ##STR333## ##STR334## ##STR335##
##STR336## ##STR337##
196-197. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present application claims benefit of provisional patent
application Ser. No. 60/474,288 filed May 28, 2003.
FIELD OF THE INVENTION
[0002] 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.sub.2) and their effect on diseases
characterized by abnormal cell mitosis and/or abnormal
angiogenesis.
BACKGROUND OF THE INVENTION
[0003] 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.
[0004] 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.
[0005] 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.
[0006] One example of a disease dependent on 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. 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, neovascular glaucoma, and retrolental 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 Sjogren'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.
[0007] Diseases associated with neovascularization include, but are
not limited to, retinal/choroidal neovascularization, 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, Best's disease, myopia, optic pits,
Stargardt's disease, pars planitis, chronic retinal detachment,
hyperviscosity syndromes, toxoplasmosis, trauma and post-laser
complications. Other 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.
[0008] Another angiogenesis associated disease is rheumatoid
arthritis. 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. Angiogenesis
may also play a role in osteoarthritis. The activation of the
chondrocytes by angiogenic-related factors contributes to the
destruction of the joint. At a later stage, the angiogenic factors
promote new bone growth. Therapeutic intervention that prevents the
cartilage destruction could halt the progress of the disease and
provide relief for persons suffering with arthritis.
[0009] Chronic inflammation may also involve pathological
angiogenesis. Such diseases as ulcerative colitis and Crohn's
disease show histological changes with the ingrowth of new blood
vessels into 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.
[0010] The hypothesis that tumor growth is angiogenesis-dependent
was first proposed in 1971. (Folkman, New Eng. J. Med., 285:1182-86
(1971)). In its simplest terms, this hypothesis states: "Once 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.
[0011] Examples of the indirect evidence which support this concept
include:
[0012] (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)).
[0013] (2) Tumors grown in isolated perfused organs where blood
vessels do not proliferate are limited to 1-2 mm.sup.3 but expand
rapidly to >1000 times this volume when they are transplanted to
mice and become neovascularized. (Folkman, et al., Annals of
Surgery, 164:491-502 (1966)).
[0014] (3) Tumor growth in the avascular cornea proceeds slowly and
at a linear rate, but switches to exponential growth after
neovascularization. (Gimbrone, Jr., et al., J. Nat. Cancer Inst.,
52:421-27 (1974)).
[0015] (4) Tumors suspended in the aqueous fluid of the anterior
chamber of a rabbit eye remain viable, avascular, and limited in
size to <1 mm.sup.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).
[0016] (5) When tumors are implanted on a chick embryo
chorioallantoic membrane, they grow slowly during an avascular
phase of >72 hours, but do not exceed a mean diameter of
0.93+0.29 mm. Rapid tumor expansion occurs within 24 hours after
the onset of neovascularization, and by day 7 these vascularized
tumors reach a mean diameter of 8.0+2.5 mm. (Knighton, British J.
Cancer, 35:347-56 (1977)).
[0017] (6) Vascular casts of metastases in a 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)).
[0018] (7) In transgenic mice that develop carcinomas in the beta
cells of the pancreatic islets, pre-vascular hyperplastic islets
are limited in size to <1 mm. At 6-7 weeks of age, 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. (Folkman, et al., Nature, 339:58-61
(1989)).
[0019] (8) A specific antibody against VEGF (vascular endothelial
growth factor) 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. (Kim,
et al., Nature, 362:841-44 (1993)).
[0020] (9) 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)).
[0021] (10) 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 tumor cells
in vitro. (Gross, et al., Proc. Am. Assoc. Cancer Res., 31:79
(1990)).
[0022] (11) 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.
[0023] (12) Human retinoblastomas that are metastatic to the
vitreous develop into avascular spheroids that are restricted to
less than 1 mm.sup.3 despite the fact that they are viable and
incorporate .sup.3H-thymidine (when removed from an enucleated eye
and analyzed in vitro).
[0024] (13) Carcinoma of the ovary metastasizes to the peritoneal
membrane as tiny avascular white seeds (1-3 mm.sup.3). These
implants rarely grow larger until one or more of them becomes
neovascularized.
[0025] (14) Intensity of neovascularization in breast cancer
(Weidner, et al., New Eng. J. Med., 324:1-8 (1991); Weidner, et
al., J Nat. Cancer Inst., 84:1875-87 (1992)) and in prostate cancer
(Weidner, et al., Am. J. Pathol., 143(2):401-09 (1993)) correlates
highly with risk of future metastasis.
[0026] (15) Metastasis from human cutaneous melanoma is rare prior
to neovascularization. The onset of neovascularization leads to
increased thickness of the lesion and an increased risk of
metastasis. (Srivastava, et al., Am. J. Pathol., 133:419-23
(1988)).
[0027] (16) In bladder cancer, the urinary level of an angiogenic
protein, bFGF, is a more sensitive indicator of status and extent
of disease than is cytology. (Nguyen, et al., J. Nat. Cancer Inst.,
85:241-42 (1993)).
[0028] Thus, it is clear that 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.
[0029] 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
that angiogenesis plays a role in the abnormalities in the bone
marrow that give rise to leukemia tumors and multiple myeloma
diseases.
[0030] 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 tumors 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.
[0031] 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.
[0032] What is needed, therefore, is a composition and method that
can inhibit angiogenesis. What is also needed is a composition and
method that can inhibit the unwanted growth of blood vessels,
especially in tumors.
[0033] 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.
[0034] In wound healing, 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.
[0035] Several compounds have been used to inhibit angiogenesis.
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. 5,001,116 and 4,994,443) have disclosed
the use of heparin and steroids to control angiogenesis. Steroids,
such as tetrahydrocortisol, which lack glucocorticoid and
mineralocorticoid activity, have been found to be angiogenic
inhibitors.
[0036] Other factors found endogenously in animals, such as 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. For example, 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)). Human recombinant interferon
(alpha/A) was reported to be successfully used in the treatment of
pulmonary hemangiomatosis, an angiogenesis-induced disease. (White,
et al., New Eng. J. Med., 320:1197-1200 (1989)).
[0037] Other agents that 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. (Japanese Kokai Tokkyo Koho No. 63-119500).
Additional anti-angiogenic compounds include Angiostatin.RTM. (U.S.
Pat. 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 Endostatin (U.S. Pat. No.
5,854,205).
[0038] 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 treat a number of 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. Hypotheses,
30(2):105-9 (1989)).
[0039] 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 that 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 dysfunction in their extremities.
[0040] Thus, improved methods and compositions are needed that are
easily administered and capable of inhibiting angiogenesis. What is
needed are safe and effective treatments that do not create
unwanted side effects.
[0041] 2-Methoxyestradiol is an endogenous metabolite of estradiol
(E2). When administered orally, it exhibits anti-tumor and
anti-proliferative activity with little toxicity. In vitro data
suggests that 2-methoxyestradiol does not engage the estrogen
receptor for its anti-proliferative activity and is not estrogenic
over a wide range of concentrations, as assayed by estrogen
dependant MCF-7 cell proliferation. However, the presence of
metabolizing enzymes, such as demethylases, in vivo and in vitro
may metabolize this compound to products, such as
2-hydroxyestradiol, which has been shown to be estrogenic by
several approaches. What is needed is a means to improve the
bioavailibility of estradiol derivatives or 2-methoxyestradiol and
to reduce the formation of estrogenic 2-methoxyestradiol
metabolites. What is also needed is a means to modify estradiol
derivatives or 2-methoxyestradiol in such a way as to prevent
conversion into an estrogenic derivative, metabolic conjugation
and/or conversion to estrones.
SUMMARY OF THE INVENTION
[0042] 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, 3 and 17
positions thereof. Compounds within the general formulae that
inhibit cell proliferation are preferred. Compounds within Formula
I 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.
[0043] A mammalian disease characterized by undesirable cell
mitosis, as defined herein, 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
Osler Weber syndrome (Osler-Weber-Rendu disease). Other undesired
angiogenesis involves normal processes including ovulation and
implantation of a blastula.
[0044] Accordingly, the compositions described above can be used to
block ovulation and implantation of a blastula or to block
menstruation (induce amenorrhea).
[0045] A novel series of compounds have been prepared that retain
the biological activities of 2ME.sub.2 but are believed to have
reduced metabolism. 17-Position alkylated analogs lack the hydroxyl
moiety and cannot be metabolized to estrones or conjugated at that
position but retain antiproliferative activity in HUVEC and tumor
cells. Replacement of the 2-methoxy group by other moieties, such
as a propynyl group, retains antiproliferative activity, but these
groups cannot be de-methylated to yield the estrogenic 2-hydroxyl
derivatives. Contrary to what is observed with 2ME.sub.2, several
of these new analogs have selective in vitro antiproliferative
activity for the endothelial cells over the tumor cell line
assessed.
[0046] Also disclosed are compounds and methods for altering the
chemical nature of positions 3 and 17 of 2-methoxyestradiol for
preventing conversion to 2-methoxyestrone and/or the conjugation of
2-methoxyestradiol (or metabolites) with other molecules and
subsequent loss during excretion of the resulting compounds.
[0047] Other features and advantages of the invention will be
apparent from the following description of preferred embodiments
thereof.
DETAILED DESCRIPTION OF THE INVENTION
[0048] As described below, compounds that are useful in accordance
with the invention include novel 2-methoxyestradiol derivatives
that exhibit anti-mitotic, anti-angiogenic and/or anti-tumor
properties. Preferred compounds of the invention are
2-methoxyestradiol derivatives modified at the 2, 3 and 17
positions. Preferred compounds are those of the general Formula
(I): ##STR2## wherein, R.sub.a is selected from --OCH.sub.3,
--OCH.sub.2CH.sub.3, or --CCCH.sub.3; Z' is selected from >C--F,
>C--NH.sub.2, >CCONH.sub.2, >C--NHCOH,
>C--OSO.sub.2NH.sub.2, or >C--CHCH.sub.2; and Z'' is selected
from >C(H.sub.2), >C(H)--CH.sub.3, >C.dbd.CH.sub.2,
>C.dbd.CHCH.sub.3, or >C.dbd.O; provided that when Z' is
>C--OSO.sub.2NH.sub.2 and Z'' is >C(H.sub.2) or >C.dbd.O,
R.sub.a is neither --OCH.sub.3 nor --OCH.sub.2CH.sub.3. Specific
compounds according to the invention are described below.
[0049] In an alternate disclosed embodiment of the present
invention, compounds according to the present invention are those
of Formula I, wherein R.sub.3 is --OCH.sub.3; Z' is selected from
>C--NH.sub.2, >CCONH.sub.2, >C--NHCOH,
>C--OSO.sub.2NH.sub.2, or >C--CHCH.sub.2; and Z'' is selected
from >C(H.sub.2), >C(H)--CH.sub.3, >C.dbd.CH.sub.2,
>C.dbd.CHCH.sub.3, or >C.dbd.O.
[0050] In another alternate disclosed embodiment of the present
invention, compounds according to the present invention are those
of Formula I, wherein R.sub.a is --OCH.sub.2CH.sub.3; Z' is
selected from >C--NH.sub.2, >CCONH.sub.2, >C--NHCOH,
>C--OSO.sub.2NH.sub.2, or >C--CHCH.sub.2; and Z'' is selected
from >C(H.sub.2), >C(H)--CH.sub.3, >C.dbd.CH.sub.2,
>C.dbd.CHCH.sub.3, or >C.dbd.O.
[0051] In a further alternate disclosed embodiment of the present
invention, compounds according to the present invention are those
of Formula I, wherein R.sub.a is --CCCH.sub.3: Z' is selected from
>C--NH.sub.2, >CCONH.sub.2, >C--NHCOH,
>C--OSO.sub.2NH.sub.2, or >C--CHCH.sub.2; and Z'' is selected
from >C(H.sub.2), >C(H)--CH.sub.3, >C.dbd.CH.sub.2,
>C.dbd.CHCH.sub.3, or >C.dbd.O.
[0052] In another alternate disclosed embodiment of the present
invention, compounds according to the present invention are those
of Formula I, wherein Z' is selected from >C--F; R.sub.a is
selected from --OCH.sub.3, --OCH.sub.2CH.sub.3, or --CCCH.sub.3;
and Z'' is selected from >C(H.sub.2), >C(H)--CH.sub.3,
>C.dbd.CH.sub.2, >C.dbd.CHCH.sub.3, or >C.dbd.O.
[0053] In another alternate disclosed embodiment of the present
invention, compounds according to the present invention are those
of Formula I, wherein Z' is >C--NH.sub.2; R.sub.a is selected
from OCH.sub.3, --OCH.sub.2CH.sub.3, or --CCCH.sub.3; and Z'' is
selected from >C(H.sub.2), >C(H)--CH.sub.3,
>C.dbd.CH.sub.2, >C.dbd.CHCH.sub.3, or >C.dbd.O.
[0054] In still another alternate disclosed embodiment of the
present invention, compounds according to the present invention are
those of Formula I, wherein Z' is selected from >CCONH.sub.2;
R.sub.a is --OCH.sub.3, --OCH.sub.2CH.sub.3, or --CCCH.sub.3; and
Z'' is selected from >C(H.sub.2), >C(H)--CH.sub.3,
>C.dbd.CH.sub.2, >C.dbd.CHCH.sub.3, or >C.dbd.O.
[0055] In a further alternate disclosed embodiment of the present
invention, compounds according to the present invention are those
of Formula I, wherein Z' is selected from >C--OSO.sub.2NH.sub.2;
R.sub.3 is --OCH.sub.3, --OCH.sub.2CH.sub.3, or --CCCH.sub.3; and
Z'' is selected from >C(H.sub.2), >C(H)--CH.sub.3,
>C.dbd.CH.sub.2, >C.dbd.CHCH.sub.3, or >C.dbd.O.
[0056] In another alternate disclosed embodiment of the present
invention, compounds according to the present invention are those
of Formula I, wherein Z' is selected from >C--CHCH.sub.2;
R.sub.a is --OCH.sub.3, --OCH.sub.2CH.sub.3, or --CCCH.sub.3; and
Z'' is selected from >C(H.sub.2), >C(H)--CH.sub.3,
>C.dbd.CH.sub.2, >C.dbd.CHCH.sub.3, or >C.dbd.O.
[0057] In yet alternate disclosed embodiment of the present
invention, compounds according to the present invention are those
of Formula I, wherein Z'' is selected from >C(H.sub.2); R.sub.a
is selected from --OCH.sub.3, --OCH.sub.2CH.sub.3, or --CCCH.sub.3;
and Z' is selected from >C--NH.sub.2, >CCONH.sub.2,
>C--NHCOH, >C--OSO.sub.2NH.sub.2, or >C--CHCH.sub.2.
[0058] In another alternate disclosed embodiment of the present
invention, compounds according to the present invention are those
of Formula I, wherein Z'' is selected from >C(H)--CH.sub.3;
R.sub.a is selected from --OCH.sub.3, --OCH.sub.2CH.sub.3, or
--CCCH.sub.3; and Z' is selected from >C--NH.sub.2,
>CCONH.sub.2, >C--NHCOH, >C--OSO.sub.2NH.sub.2, or
>C--CHCH.sub.2.
[0059] In a further alternate disclosed embodiment of the present
invention, compounds according to the present invention are those
of Formula I, wherein Z'' is selected from >C.dbd.CH.sub.2;
R.sub.a is selected from --OCH.sub.3, --OCH.sub.2CH.sub.3, or
--CCCH.sub.3; and Z' is selected from >C--NH.sub.2,
>CCONH.sub.2, >C--NHCOH, >C--OSO.sub.2NH.sub.2, or
>C--CHCH.sub.2.
[0060] In another alternate disclosed embodiment of the present
invention, compounds according to the present invention are those
of Formula I, wherein Z'' is selected from >C.dbd.CHCH.sub.3;
R.sub.a is selected from OCH.sub.3, --OCH.sub.2CH.sub.3, or
--CCCH.sub.3; and Z' is selected from >C--NH.sub.2,
>CCONH.sub.2, >C--NHCOH, >C--OSO.sub.2NH.sub.2, or
>C--CHCH.sub.2.
[0061] In still another alternate disclosed embodiment of the
present invention, compounds according to the present invention are
those of Formula I, wherein Z'' is selected from >C.dbd.O;
R.sub.a is selected from --OCH.sub.3, --OCH.sub.2CH.sub.3, or
--CCCH.sub.3; and Z' is selected from >C--NH.sub.2,
>CCONH.sub.2, >C--NHCOH, >C--OSO.sub.2NH.sub.2, or
>C--CHCH.sub.2.
[0062] In a further alternate disclosed embodiment of the present
invention, compounds according to the present invention are those
of Formula I, wherein R.sub.a is selected from --OCH.sub.3,
--OCH.sub.2CH.sub.3, or --CCCH.sub.3; Z' is selected from
>C--NH.sub.2, >CCONH.sub.2, >C--NHCOH, or
>C--CHCH.sub.2; and Z'' is selected from >C(H.sub.2),
>C(H)--CH.sub.3, >C.dbd.CH.sub.2, >C.dbd.CHCH.sub.3, or
>C.dbd.O.
[0063] In another alternate disclosed embodiment of the present
invention, compounds according to the present invention are those
of Formula I, wherein R.sub.a is selected from --OCH.sub.3,
--OCH.sub.2CH.sub.3, or --CCCH.sub.3; Z' is selected from
>C--NH.sub.2, >CCONH.sub.2, >C--NHCOH,
>C--OSO.sub.2NH.sub.2, or >C--CHCH.sub.2; and Z'' is selected
from >C(H.sub.2), >C(H)--CH.sub.3, >C.dbd.CH.sub.2,
>C.dbd.CHCH.sub.3, or >C.dbd.O; provided that when Z' is
>C--OSO.sub.2NH.sub.2, Z'' is neither >C(H.sub.2) nor
>C.dbd.O.
[0064] 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.
[0065] Although not wishing to be bound by theory, it is believed
that 2-methoxyestrone (2ME.sub.1) is formed through the same
enzymatic pathway as estrone is formed from estradiol. Although not
wishing to be bound by theory, it is further believed that the
enzymes responsible for this reaction on estradiol are the
17.beta.-hydroxysteroid dehydrogenases (17.beta.-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). Each
of the four members of this enzyme family, types 1, 2, 3, and 4
have distinct activity. It appears that 17.beta.-HSD type 1
catalyzes the reductive reaction (estrone to estradiol), while
17.beta.-HSD type 2 catalyzes the oxidation reaction (estradiol to
estrone), and type 3 catalyzes 4-androstenedione to testosterone.
It is also believed that an additional metabolic deactivation
pathway results in conjugation of 2-methoxyestradiol or
2-methoxyestrone with molecules such as sulfate or glucuronic acid
and subsequent loss via excretion. In this invention, positions 3
and 17 of 2-methoxyestradiol, and derivatives thereof, may be
modified to prevent these metabolic pathways from occurring.
[0066] Since 2-methoxyestradiol is metabolized to a much less
active metabolite, the present invention adds steric bulk and/or
modification of chemical or electrostatic characteristics at
positions 3 and 17 of 2-methoxyestradiol for retarding or
preventing interaction of the family of 17.beta.-hydroxysteroid
dehydrogenases and co-factor NADP.sup.+ on this substrate. Addition
of steric bulk and/or modification of chemical or electrostatic
characteristics at positions 3 and 17 of 2-methoxyestradiol may
also retard or prevent conjugation, such as glucuronidation or
sulfation. It is believed that retardation or prevention of these
two metabolic deactivation pathways prolongs the serum lifetime of
2-methoxyestradiol and other estradiol derivatives while retaining
the desired anti-angiogenic and anti-tumor activity.
[0067] Aside from preventing the possible metabolism of 2ME.sub.2
to 2ME.sub.1, which may occur by making these steroids poor
substrates for 17.beta.-HSD (by either steric and/or electronic
effects), it is not possible for some of these analogs modified at
the 2 position to undergo the demethylation known to occur with
2ME.sub.2 since there is no methyl ether group at that position.
This is desirable since it has been demonstrated that
2-hydroxyestradiol (the product of demethylation of 2ME.sub.2) has
estrogenic activity.
[0068] It is well known that orally-delivered steroids, such as
estradiol (E.sub.2) and ethynyl-E.sub.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,
R.; Nawreth. P. P.; Schweigerer, L., The Endogenous Estrogen
Metabolite 2-Methoxyestradiol Inhibits Angiogenesis and Suppresses
Tumor. Nature, 1994, 368, 237-239; Wang, Z.; Yang. D.;
Mohanakrishnan, A. K.; Fanwick, P. E.; Nampoothiri, P.; Hamel, E.;
Cushman, M. "Synthesis of B-Ring Homologated Estradiol Analogs that
Modulate Tubulin Polymerization and Microtubule Stability." J. Med.
Chem., 2000, 43, 2419-2429) e.g. oxidation at the D-ring's
17-hydroxy group of E.sub.2 to form estrone and conjugation with
sulfate and/or glucuronate at the hydroxyls of position-3 on the
A-ring and position-17 on the D-ring.
[0069] Several studies have been conducted to determine SAR of
2ME.sub.2 analogs (D'Amato, R. J.; Lin, C. M.; Flynn, E.; Folkman,
J.; Hamel, E. Inhibition of Angiogenesis and Breast Cancer in Mice
by the Microtubule Inhibitors 2-Methoxyestradiol and Taxol", Cancer
Res., 1997, 57, 81-86; Cushman, M.; He, M.-H.; Katzenellenbogen, J.
A.; Lin, C. M.; Hamel, E. "Synthesis, Antitubuln and Antimitotic
Activity, and Cytotoxicity of Analogs of 2-Methoxyestradiol, an
Endogenous Mammalian Metabolite of Estradiol that inhibits Tubulin
Polymerization by Binding to the Colchicine Binding Site." J. Med.
Chem. 1995, 38, 2041-2049) but none to reduce or stop its metabolic
pathway.
[0070] In the preferred embodiment of the invention,
2-methoxyestradiol, and derivatives thereof, are modified at the 3
and 17 positions.
Anti-Proliferative Activity In Situ
[0071] Anti-proliferative activity can be 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. Pat. No. 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 .mu.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/or angiogenesis.
Anti-Proliferative Activity In Vitro
[0072] The process by which 2ME.sub.2 affects cell growth remains
unclear, however, a number of studies have implicated various
mechanisms of action and cellular targets. 2ME.sub.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.,
Viljoen, T. C., Grobler, C. J. S. and Seegers, J. C. (1996)
17.beta.-Estradiol metabolites affect some regulators of the MCF-7
cell cycle. Cancer Letters 110, 181-186); cell division cycle
kinases and regulators, e.g., p34.sup.cdc2 and cyclin B (Lottering
et al. (1996); Attalla, H., Makela, T. P., Adlercreutz, H. and
Andersson, L. C. (1996) 2-Methoxyestradiol arrests cells in mitosis
without depolymerizing tubulin. Biochemical and Biophysical
Research Communications 228, 467-473; Zoubine, M. N., Weston, A.
P., Johnson, D. C., Campbell, D. R. and Banerjee, S. K. (1999)
2-Methoxyestradiol-induced growth suppression and lethality in
estrogen-responsive MCF-7 cells may be mediated by down regulation
of p34cdc2 and cyclin B1 expression. Int J Oncol 15, 639-646);
transcription factor modulators, e.g., SAPK/JNK (Yue, T-L., Wang,
X., Louden, C. S., Gupta, L. S., Pillarisetti, K., Gu, J-L., Hart,
T. K., Lysko, P. G. and Feuerstein, G. Z. (1997)
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. Molecular Pharmacology 51, 951-962; Attalla, H.,
Westberg, J. A., Andersson, L. C., Aldercreutz, H. and Makela, T.
P. (1998) 2-Methoxyestradiol-induced phosphorylation of bcl-2:
uncoupling from JNK/SAPK activation. Biochem and Biophys Res Commun
247, 616-619); and regulators of cell arrest and apoptosis, e.g.,
tubulin (D'Amato, R. J., Lin, C. M., 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, C. M., Flynn, E. and D'Amato, R. J. (1996) Interactions of
2-methoxyestradiol, and endogenous mammalian metabolite, with
unpolymerized tubulin and with tubulin polymers. Biochemistry 35,
1304-1310), p.sub.21.sup.WAF1/CIP1 (Mukhopadhyay, T. and Roth, J.
A. (1997) Induction of apoptosis in human lung cancer cells after
wild-type p53 activation by methoxyestradiol. Oncogene 14,
379-384), bcl-2 and FAS (Yue et al. (1997); Attalla et al. (1998)),
and p53 (Kataoka, M., Schumacher, G., Cristiano, R. J., Atkinson,
E. N., Roth, J. A. and Mukhopadhyay, T. (1998) An agent that
increases tumor suppressor transgene product coupled with systemic
transgene delivery inhibits growth of metastatic lung cancer in
vivo. Cancer Res 58, 4761-4765; Mukhopadhyay et al. (1997);
Seegers, J. C., Lottering, M-L., Grobler C. J. S., van Papendorp,
D. H., Habbersett, R. C., Shou, Y. and Lehnert B. E. (1997) The
mammalian metabolite, 2-methoxyestradiol, affects p53 levels and
apoptosis induction in transformed cells but not in normal cells.
J. Steroid Biochem. Molec. Biol. 62, 253-267). The effects on the
level of cAMP, calmodulin activity and protein phosphorylation may
also be related to each other. More recently, 2ME.sub.2 was shown
to upregulate Death Receptor 5 and caspase 8 in human endothelial
and tumor cell lines (LaVallee T M, Zhan X H, Johnson M S,
Herbstritt C J, Swartz G, Williams M S, Hembrough W A, Green S J,
Pribluda V S. 2-methoxyestradiol up-regulates death receptor 5 and
induces apoptosis through activation of the extrinsic pathway.
Cancer Res. (2003) 63#2:468-75). Additionally, 2ME.sub.2 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.). All cellular targets described above are not
necessarily mutually exclusive to the inhibitory effects of
2ME.sub.2 in actively dividing cells.
[0073] The high affinity binding of 2ME.sub.2 to SHBG has been
mechanistically associated to its efficacy in a canine model of
prostate cancer, in which signaling by estradiol and
5.alpha.-androstan-3.alpha.,17.beta.-diol were inhibited by
2ME.sub.2 (Ding, V. D., Moller, D. E., Feeney, W. P., Didolkar, V.,
Nakhla, A. M., Rhodes, L., Rosner, W. and Smith, R. G., Sex
hormone-binding globulin mediates prostate androgen receptor action
via a novel signaling pathway, Endocrinology 139, 213-218
(1998)).
[0074] The more relevant mechanisms described above have been
extensively discussed in Victor S. Pribluda, Theresa M. LaVallee
and Shawn J. Green, 2-Methoxyestradiol: A novel endogenous
chemotherapeutic and antiangiogenic in The New Angiotherapy,
Tai-Ping Fan and Robert Auerbach eds., Human Press Publisher.
[0075] Assays relevant to these mechanisms of action and inhibition
of cell proliferation are well-known in the art. For example,
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 can be followed in a
Gilford recording spectrophotometer (model 250 or 2400S) equipped
with electronic temperature controllers. A reaction mixture
typically contains 1.0 M monosodium glutamate (pH 6.6), 1.0 mg/ml
(10 .mu.M) tubulin, 1.0 mM MgCl.sub.2, 4% (v/v) dimethylsulfoxide
and 20-75 .mu.M of a composition to be tested. The reaction
mixtures are incubated for 15 min. at 37.degree. C. and then
chilled on ice. After addition of 10 .mu.l 2.5 mM GTP, the reaction
mixture is transferred to a cuvette at 0.degree. C., and a baseline
established. At time zero, the temperature controller of the
spectrophotometer is set at 37.degree. C. Microtubule assembly is
evaluated by increased turbity at 350 nm. Alternatively, inhibition
of microtubule assembly can be followed by transmission electron
microscopy as described in Example 2 of U.S. Pat. Nos. 5,504,074,
5,661,143, and 5,892,069, the disclosures of which are incorporated
herein by reference.
[0076] Other such assays include counting of cells in tissue
culture plates or assessment of cell number through metabolic
assays or incorporation into DNA of labeled (radiochemically, for
example .sup.3H-thymidine, or fluorescently labeled) or
immuno-reactive (BrdU) nucleotides. In addition, 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.
Indications
[0077] The invention can be used to treat any disease characterized
by abnormal cell mitosis and/or abnormal angiogenesis. Such
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, 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,
neuroscular glaucoma, liver diseases and Oster Webber syndrome
(Osler-Weber Rendu disease).
[0078] Diseases associated with neovascularization can be treated
according to the present invention. Such diseases include, but are
not limited to, diabetic retinopathy, retinopathy of prematurity,
corneal graft rejection, neovascular glaucoma and retrolental
fibroplasias, epidemic keratoconjunctivitis, Vitamin A deficiency,
contact lens overwear, atopic keratitis, superior limbic keratitis,
pterygium keratitis sicca, Sjogren's, 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, marginal keratolysis, trauma, rheumatoid arthritis,
systemic lupus, polyarteritis, Wegener's sarcoidosis, Scleritis,
Steven-Johnson disease, pemphigoid, radial keratotomy, and corneal
graph rejection.
[0079] Other diseases associated with neovascularization can be
treated according to the present invention. Such diseases 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, Bechet'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.
Other 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.
[0080] The present invention may also be used to treat cancerous
diseases. Cancerous diseases include, but are not limited to,
rhabdomyosarcoma, retinoblastoma, Ewing sarcoma, neuroblastoma,
osteosarcoma, acoustic neuromas, neurofibromas, hemangiomas, breast
cancer, prostrate cancer, renal cell cancer, brain tumors, ovarian
cancer, colon cancer, bladder cancer, cutaneous melanoma, liver
cancer, and lung cancer.
[0081] Another disease that 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.
[0082] Other diseases that can be treated according to the present
invention are hemangiomas, Osler-Weber-Rendu disease, or hereditary
hemorrhagic telangiectasia, solid or blood borne tumors and
acquired immune deficiency syndrome.
[0083] In addition, 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.
[0084] Also contemplated by the present invention are implants or
other devices comprised of the compounds or drugs of Formula I or
prodrugs thereof where the drug or prodrug is formulated in a
bio-degradable or non-biodegradable polymer for sustained release.
Non-biodegradable polymers release the drug in a controlled fashion
through physical or mechanical processes without the polymer itself
being degraded. Biodegradable polymers are designed to gradually be
hydrolyzed or solubilized by natural processes in the body,
allowing gradual release of the admixed drug or prodrug. Both
bio-degradable and non-biodegradable polymers and the process by
which drugs are incorporated into the polymers for controlled
release are well known to those skilled in the art. Examples of
such polymers can be found in many references such as Brem et al,
J. Neurosurg 74: pp. 441-446 (1991). These implants or devices can
be implanted in the vicinity where delivery is desired, for
example, at the site of a tumor or a stenosis.
[0085] Because anything not formed in the body as a natural
component may elicit extreme and unexpected responses, such as
blood vessel closure due to thrombus formation or spasm, and
because damage to blood vessels by the act of insertion of a
vascular stent may be extreme and unduly injurious to the blood
vessel surface, it is prudent to protect against such events.
Restenosis is a re-narrowing or blockage of an artery at the same
site where treatment, such as an angioplasty or stent procedure,
has already taken place. If restenosis occurs within a stent that
has been placed in an artery, it is technically called "in-stent
restenosis," the end result being a narrowing in the artery caused
by a build-up of substances that may eventually block the flow of
blood. The compounds that are part of the present invention are
especially useful to coat vascular stents to prevent restenosis.
The coating should preferably be a biodegradable or
non-biodegradable polymer that allows for a slow release of a
compound of the present invention thereby preventing the restenosis
event.
Prodrug
[0086] The present invention also relates to conjugated prodrugs
and uses thereof. More particularly, the invention relates to
conjugates of estradiol compounds such as compounds of Formula I,
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.
[0087] In one aspect, the present invention provides a conjugated
prodrug of an estradiol compound, preferably compounds of Formula
I, conjugated to a biological activity modifying agent.
[0088] Alternatively, the conjugated prodrug according to the
present invention includes the compounds of Formula I conjugated to
a peptide moiety.
[0089] The incorporation of an estradiol compound, such as the
compounds of Formula I, into a disease-dependently activated
pro-drug enables significant improvement of potency and selectivity
of this anti-cancer and anti-inflammatory agent.
[0090] In addition to the compounds of the present invention, 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.
[0091] In addition, the compounds of Formula I or prodrug thereof
may be incorporated into biodegradable or non-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. Polymers and their use are
described in detail in Brem et al., J. Neurosurg 74:441-446
(1991).
[0092] A person skilled in the art will be able by reference to
standard texts, such as Remington's Pharmaceutical Sciences 17th
edition, to determine how the formulations are to be made and how
these may be administered.
[0093] In a further aspect of the present invention there is
provided use of compounds of Formula I or prodrugs thereof
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.
[0094] In a further aspect of the present invention there is
provided a pharmaceutical composition comprising compounds of
Formula I or prodrugs thereof according to the present invention,
together with a pharmaceutically acceptable carrier, diluent or
excipient.
[0095] The pharmaceutical composition may be used for the
prophylaxis or treatment of conditions associated with angiogenesis
or accelerated cell division or inflammation.
[0096] In a still further aspect of the present invention there is
provided a method of prophylaxis or treatment of a condition
associated with angiogenesis or accelerated or increased amounts of
cell division hypertrophic growth or inflammation, said method
including administering to a patient in need of such prophylaxis or
treatment an effective amount of compounds of Formula I or prodrugs
thereof according to the present invention, as described above.
[0097] It should be understood that prophylaxis or treatment of
said condition includes amelioration of said condition.
[0098] By "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.
[0099] 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.
Improved Estradiol Derivative Synthesis
[0100] Known compounds that are used in accordance with the
invention and precursors to novel compounds according to the
invention can be purchased, e.g., from Sigma Chemical Co., St.
Louis, Steraloids or Research Plus. Other compounds according to
the invention can be synthesized according to known methods from
publicly available precursors.
[0101] The chemical synthesis of estradiol has been described
(Eder, V. et al., Ber 109, 2948 (1976); Oppolzer, D. A. and
Roberts, D A. 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 (Trembley et al., Bioorganic & Med. Chem.
1995 3, 505-523; Fevig et al., J. Org. Chem., 1987 52, 247-251;
Gonzalez et al., Steroids 1982, 40, 171-187; Trembley et al.,
Synthetic Communications 1995, 25, 2483-2495; Newkome et al., J.
Org. Chem. 1966, 31, 677-681; Corey et al Tetrahedron Lett 1976,
3-6; Corey et al., Tetrahedron Lett, 1976, 3667-3668) and German
Patent No. 2757157 (1977).
Administration
[0102] The compositions described above can be provided as
physiologically acceptable formulations using known techniques, and
these formulations can be administered by standard routes. In
general, the combinations may be administered by the topical, oral,
rectal or parenteral (e.g., intravenous, subcutaneous or
intramuscular) route. In addition, the combinations may be
incorporated into 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 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, especially 0.01-20
mg/kg/day, is generally preferred.
[0103] The formulations include those suitable for oral, rectal,
nasal, inhalation, topical (including dermal, transdermal, buccal
and sublingual), vaginal or parenteral (including subcutaneous,
intramuscular, intravenous, intradermal, intraocular,
intratracheal, and epidural) and inhalation 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 a pharmaceutical carrier(s) or excipient(s). In
general, the formulations are prepared by uniformly and intimately
bringing into association the active ingredient with liquid
carriers or finely divided solid carriers or both, and then, if
necessary, shaping the product.
[0104] 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, etc.
[0105] 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.
[0106] 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.
[0107] 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.
[0108] Formulations for rectal administration may be presented as a
suppository with a suitable base comprising, for example, cocoa
butter or a salicylate.
[0109] Formulations suitable for nasal administration, wherein the
carrier is a solid, 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.
[0110] 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, ingredients such as carriers as are known in the art to
be appropriate.
[0111] Formulation suitable for inhalation may be presented as
mists, dusts, powders or spray formulations containing, in addition
to the active ingredient, ingredients such as carriers as are known
in the art to be appropriate.
[0112] 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 tablets of the kinds previously described.
[0113] 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.
[0114] 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.
[0115] The present invention includes compositions and methods for
treating mammalian disease characterized by pathogenic angiogenesis
by administering compounds of Formula I. The 2-methoxyestradiol,
and derivatives thereof, are modified at the 3 and 17 positions.
Combinations which are physically impossible are not contemplated
by this invention, such as a carbon atom containing 5 bonds.
[0116] 100% pure isomers are contemplated by this invention,
however a stereochemical isomer labeled as .alpha. or .beta. may be
a mixture of both in any ratio, where it is chemically possible by
one skilled in the art. Also contemplated by this invention are
both classical and non-classical bioisosteric atom and substituent
replacements, such as are described by Patani and Lavoie
(Bio-isosterism: a rational approach in drug design Chem. Rev.
(1996) p. 3147-3176) and are well known to one skilled in the art.
Such bioisosteric replacements include, for example, substitution
of .dbd.S or .dbd.NH for .dbd.O.
[0117] The synthetic routes for this series of analogs are
summarized in Schemes 1-7. Schemes 1 and 2 present the preparation
of the singly and doubly substituted templates that are required as
precursors for the triply substituted analogs. Schemes 3-7 present
on the modification of position 3 of the templates derived from
Schemes 1 and 2. These synthetic routes present one potential way
to prepare this series of analogs, and other synthetic routes
(including modifying the order of synthetic steps or reagents) are
possible to someone skilled in the art.
[0118] In Scheme 1, the 2-methoxy, 2-ethoxy or 2-propynyl
derivatives, which are either commercially available or can be
readily prepared by literature methods (Wang et al, Synthetic Comm
1998, 28, 4431, Cushman et al J. Med. Chem. 1995, 38, 2041, Cushman
et al J. Med. Chem. 1997, 40, 2323, Cushman et al J. Med. Chem.
2002, 45, 4748), are oxidized using the Oppenauer oxidation. The
resulting ketone can be deoxygenated using the Wolf-Kishner
reduction (Shapiro R, J. et al J. Org Chem. 1964, 86, 2825-2832.)
or olefinated (Schow et al J. Org. Chem. 1979, 44, 22. Irubner et
al J. Org. Chem. 1968, 33, 1715.) using the Wittig reaction. Both
the 17-methylene and 17-ethylene estrane analogs can be reduced to
the alkane using catalytic reduction.
[0119] In specific cases, the nature of protecting groups or the
order of reactions may have to be altered to reach the desired
products. These changes to the general synthetic schemes would be
well understood to one skilled in the art. For instance, in the
case where the desired 2 functionality is a propyne and the desired
17-functionality is an alkyl group, catalytic hydrogenation could
not be carried out on the 17-olefin since the 2-alkyne would also
be reduced. Scheme 2 presents a synthetic route to prepare analog
18. In this example, the 17 methyl is introduced as in Scheme 1
starting with estrone, and subsequently the 2-propyne is
incorporated using a literature method (Cushman et al J. Med. Chem.
2002, 45, 4748).
[0120] Schemes 3-7 feature the further modification of the
templates generated in Schemes 1 and 2. In Scheme 3, templates 4, 5
or 6 can be converted to the 3-sulfamate derivative (19, 20 or 21)
with sulfamoyl chloride and either sodium hydride (Howarth et al J.
Med. Chem. 1994, 37, 219) or 2,6-di-tert-butyl-4-methylpyridine
(Coibanu et al J. Med. Chem. 1999, 42, 2280). To prepare analogs
22-24, triflic anhydride was added to a solution of 4, 5 or 6 in
dichloromethane and pyridine at 0.degree. C. using Echavarren's
procedure (Echvarren et al J. Am. Chem. 1987, 109, 5478). The
triflate is a versatile synthetic intermediate and was used to
incorporate a wide range of functional groups at position 3. One
utility of the triflate is its conversion to the vinyl analog via
palladium catalyized substitution with a vinyl functionality
(analogs 25-27) (Shi et al J. Med Chem. 2002, 124, 6921). The
carboxylic acid derivatives (28-30) were also prepared by palladium
catalysis as well (Shi et al Chem. & Biol. 2001, 8, 501). The
carboxylic acid could also be converted to the corresponding amide
using thionyl chloride and ammonia gas (Tomas de, Paulis, et. at.
J. Med. Chem. 1986, 29, 61). The triflate could be converted
directly to a carboxamide using a procedure described by Morera
(Tetrahedron Letters, 1998, 39, 2835-2838). Alternatively, the
triflate could be converted to an amine via palladium catalyzed
substitution of the triflate with benzophenone imine, subsequent
hydrolysis yielded the corresponding amine (analogs 34-36) (Wolfe
et al Tetrahedron Lett 1997, 38, 6367). The 3-amine could be
converted to 3-fluoro analogs using the diazonium salt intermediate
(Morrow J. Med. Chem. 1966, 9, 249). Additionally, ketones 34-36
can be reduced to the corresponding alcohols 37-39 with lithium
aluminum hydride at -78.degree. C. then converted to amides 43-45
with trimethylacetic formyl anhydride (Vleitstra et al Recueil
1982, 101, 460). Schemes 4-7 use analogous chemistry to prepare
other triply modified steroid analogs.
[0121] Compounds according to the present invention may be prepared
using the reaction schemes shown below: ##STR3## ##STR4## ##STR5##
##STR6## ##STR7## ##STR8## ##STR9##
EXPERIMENTAL DATA
[0122] The following Examples refer to compounds of the following
general Formula I: ##STR10##
[0123] wherein R.sub.a is selected from --OCH.sub.3,
--OCH.sub.2CH.sub.3 or --CCCH.sub.3; Z' is selected from >C--F,
>C--NH.sub.2, >CCONH.sub.2, >C--NHCOH,
>C--OSO.sub.2NH.sub.2 or >C--CHCH.sub.2, and Z'' is selected
from >C(H.sub.2), >C(H)--CH.sub.3, >C.dbd.CH.sub.2,
>C.dbd.CHCH.sub.3 or >C.dbd.O; provided that when Z' is
>C--OSO.sub.2NH.sub.2 and Z'' is >C(H.sub.2) or >C.dbd.O,
R.sub.a is neither --OCH.sub.3 nor --OCH.sub.2CH.sub.3. Preferred
species from the foregoing genus that are useful in the present
invention include, but are not limited to, the compounds shown in
Table I. TABLE-US-00001 TABLE I 3-Amines ##STR11## ##STR12##
##STR13## ##STR14## ##STR15## ##STR16## ##STR17## ##STR18##
##STR19## ##STR20## ##STR21## ##STR22## ##STR23## ##STR24##
##STR25## 3-Carbamides ##STR26## ##STR27## ##STR28## ##STR29##
##STR30## ##STR31## ##STR32## ##STR33## ##STR34## ##STR35##
##STR36## ##STR37## ##STR38## ##STR39## ##STR40## 3-Formamides
##STR41## ##STR42## ##STR43## ##STR44## ##STR45## ##STR46##
##STR47## ##STR48## ##STR49## ##STR50## ##STR51## ##STR52##
##STR53## ##STR54## ##STR55## 3-Sulfamates ##STR56## ##STR57##
##STR58## ##STR59## ##STR60## ##STR61## ##STR62## ##STR63##
##STR64## ##STR65## ##STR66## 3-Vinyls ##STR67## ##STR68##
##STR69## ##STR70## ##STR71## ##STR72## ##STR73## ##STR74##
##STR75## ##STR76## ##STR77## ##STR78## ##STR79## ##STR80##
##STR81## 3-Fluoro ##STR82## ##STR83## ##STR84## ##STR85##
##STR86## ##STR87## ##STR88## ##STR89## ##STR90## ##STR91##
##STR92## ##STR93## ##STR94## ##STR95## ##STR96##
[0124] Each of the foregoing compounds from Table I are found to
have anti-mitotic, anti-angiogenic and/or anti-tumor
properties.
[0125] In an alternate disclosed embodiment of the present
invention, preferred species from the foregoing genus that are
useful in the present invention include, but are not limited to,
the compounds shown in Table II. TABLE-US-00002 TABLE II ##STR97##
##STR98## ##STR99## ##STR100## ##STR101## ##STR102## ##STR103##
##STR104## ##STR105## ##STR106## ##STR107## ##STR108## ##STR109##
##STR110## ##STR111## ##STR112## ##STR113## ##STR114## ##STR115##
##STR116## ##STR117## ##STR118## ##STR119## ##STR120## ##STR121##
##STR122## ##STR123## ##STR124## ##STR125## ##STR126## ##STR127##
##STR128## ##STR129## ##STR130## ##STR131## ##STR132## ##STR133##
##STR134## ##STR135## ##STR136## ##STR137## ##STR138## ##STR139##
##STR140## ##STR141## ##STR142## ##STR143## ##STR144## ##STR145##
##STR146## ##STR147## ##STR148## ##STR149## ##STR150## ##STR151##
##STR152## ##STR153## ##STR154## ##STR155## ##STR156## ##STR157##
##STR158## ##STR159## ##STR160## ##STR161## ##STR162## ##STR163##
##STR164## ##STR165## ##STR166## ##STR167## ##STR168## ##STR169##
##STR170## ##STR171## ##STR172## ##STR173## ##STR174## ##STR175##
##STR176## ##STR177## ##STR178## ##STR179## ##STR180##
[0126] In another alternate disclosed embodiment of the present
invention, preferred species from the foregoing genus that are
useful in the present invention include, but are not limited to,
the compounds shown in Table III. TABLE-US-00003 TABLE III
##STR181## ##STR182## ##STR183## ##STR184## ##STR185## ##STR186##
##STR187## ##STR188## ##STR189## ##STR190## ##STR191## ##STR192##
##STR193## ##STR194## ##STR195## ##STR196## ##STR197## ##STR198##
##STR199## ##STR200## ##STR201## ##STR202## ##STR203## ##STR204##
##STR205## ##STR206## ##STR207## ##STR208## ##STR209## ##STR210##
##STR211## ##STR212## ##STR213## ##STR214## ##STR215## ##STR216##
##STR217## ##STR218## ##STR219## ##STR220## ##STR221## ##STR222##
##STR223## ##STR224## ##STR225## ##STR226## ##STR227## ##STR228##
##STR229## ##STR230## ##STR231## ##STR232## ##STR233## ##STR234##
##STR235## ##STR236## ##STR237## ##STR238## ##STR239##
##STR240##
EXAMPLE 1
[0127] The procedures described below are for specific compounds.
However, these reactions can be applied to all examples in this
patent by one skilled in the art. References to compound #s below
correspond to the numbers assigned to the compounds shown in the
synthesis Schemes 1-7 above.
[0128] Representative oxidation of estradiol analogs to estrone
analogs: 2-ethoxyestra-1,3,5(10)-trien-3-ol-17-one (Compound #5):
2-Ethoxyestra-1,3,5(10)-trien-3,17-diol (#2, 1.88 g, 5.67 mmol) was
placed in a 250 mL round bottom flask that was equipped with a 25
mT Dean-Stark trap and a reflux condenser. The entire apparatus had
been flame dried under an argon atmosphere. Toluene (50 mL) was
added to dissolve the starting material. Aluminum isopropoxide (5.7
g, 28.4 mmol) and cyclohexanone (23.5 mL, 2.26.8 mmol) were added
and the entire reaction mixture was heated at reflux
(145-150.degree. C.) for 20 h. Saturated aqueous sodium bicarbonate
solution (100 mL) was added after the reaction mixture was allowed
to cool to room temperature. The organic material was extracted
with dichloromethane (3.times.150 mL). The aqueous emulsion was
acidified with 3 N HCl (.about.20 mL) until the emulsion separated
and again, the aqueous layer was extracted with ethyl acetate
(2.times.75 mL). The combined organic extracts were dried over
magnesium sulfate and condensed in vacuo. The crude product was
purified via flash chromatography (silica gel, hexanes ethyl
acetate 8:1 by volume) to produce #5 as a white solid (1.7 g, 94%).
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 6.78 (s, 1H), 6.66 (s,
1H), 5.49 (s, 1H), 4.07 (dq, J=6.9 and 1.6 Hz, 2H), 2.83 (m, 2H),
2.52 (m, 2H), 2.36-1.37 (m, 11H), 1.42 (t, J=7 Hz, 3H), 0.92 (s,
3H).
[0129] Synthesis of 2-methoxyestra-1,3,5(10)-triene-3-ol (Compound
#7): Into a stirring suspension of
2-methoxyestra-1,3,5(10)-trien-3-ol-17-one (#4, 8.1 g, 30 mmol) in
diethylene glycol (60 mL), 1-butanol (20 mL) and hydrazine
anhydrous (2 mL, 60 mmol) were added. The reaction mixture was
heated under reflux for 1 hour and the solution cleared. After
cooling reaction mixture to 50.degree. C., KOH pellets (5.04 g, 90
mmol) were added and the butanol was distilled. The reaction
mixture was heated at 50.degree. C. for 2 hours and then cooled to
room temperature. After the mixture was poured onto ice (50 g), 6N
HCl (20 mL) was added with stirring to give white solid product.
The product was separated by filtration, washed with cold water and
dried under vacuum to give 7.5 g (90%) product. The product was
purified on silica gel column eluted with CHCl.sub.3/MeOH 99:1.
.sup.1H NMR in CDCl.sub.3 confirmed the product as
2-methoxyestra-1,3,5(10)-triene-3-ol (7).
[0130] Representative procedure for preparation of
17-olefin-2-alkoxyestrane or 17-olefin-2-alkylestrane
analogs--2-methoxy-17(20)-methyleneestra-1,3,5(10)-triene-3-ol
(Compound #10): Potassium-tert-amylate (1.54 M, toluene, 4.35 mL
6.69 mmol, prepared as in Schow et al J. Org. Chem. 1979, 44, 3760)
was added to a suspension of methyl triphenylphosphonium bromide
(2.39 g, 6.69 mmol) in anhydrous benzene and refluxed for 30 min.
2-Methoxyestrone (#4, 300 mg, 1 mmol) in warm benzene (5 mL) was
added and the mixture was refluxed for 3 h. The reaction was cooled
to room temperature, poured into 100 mL water, washed with ether
(2.times.100 mL). The combined organics were washed with 6 M HCl
(1.times.100 mL), NaHCO.sub.3 (saturated, 1.times.100 mL), water
(1.times.100 mL), and brine (1.times.100 mL). Dry with sodium
sulfate, filter and rotoevap to give a semi solid-yellowish oil.
Purify by silica gel column chromatography using 95:5
chloroform:methanol as an eluent. Obtain 220 mg
2-methoxy-17(20)-methyleneestra-1,3,5(10)-triene-3-ol (#10, 0.738
mmol, 73% yield). Selected spectral data: .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. 6.83 (s, 1H), 6.67 (s, 1H), 5.44 (br s, 1H),
4.70 (t, J=2.26 Hz, 2H), 3.89 (s, 3H), 2.86-2.74 (m, 2H), 2.64-2.49
(m, 1H), 2.39-2.17 (m, 3H), 2.02-1.78 (m, 3H), 1.65-1.19 (m, 6H),
0.85 (s, 3H). .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 162.2,
144.9, 143.8, 132.3, 130.0, 115.0, 108.5, 101.2, 77.6, 56.5, 53.9,
44.7, 39.2, 36.2, 29.9, 29.5, 28.1, 27.3, 24.3, 19.0. Analytical
(C.sub.20H.sub.26O.sub.2); calculated C=80.48, H=8.79; found
C=80.60, H=8.77.
[0131] 2-Methoxy-19-norpregna-1,3,5(10)17(20)-tetraene-3-ol
(Compound #13): Reaction conditions in general same as in
preparation of #10 except 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. Selected spectral data: .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 6.82 (s, 1H), 6.67 (s, 1H), 5.44 (s, 1H), 5.23-5.07 (m,
1H), 3.88 (s, 3H), 2.86-2.72 (m, 2H), 2.51-2.38 (m, 2H), 2.38-2.17
(m, 3H), 1.99-1.88 (m, 1H), 1.83-1.68 (m, 4H), 1.49-1.20 (m, 6H),
0.94 (s, Z isomer) and 0.80 (s, E isomer, total 3H, ratio 5:1
respectively). .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 153.0 (E
isomer) and 150.7 (Z isomer), 145.0, 143.8, 132.4, 130.0, 115.0,
113.8, 110.6 (Z isomer) and 108.4 (E isomer), 56.5, 55.6, 54.1,
45.0 (Z isomer) and 44.5 (E isomer), 39.0 (E isomer) and 38.7 (Z
isomer), 37.7 (Z isomer) and 36.6 (E isomer), 31.9, 29.5, 28.1 (E
isomer) and 28.0 (Z isomer), 27.7 (Z isomer) and 27.4 (E isomer),
24.5 (Z isomer) and 24.4 (E isomer), 19.5 (E isomer) and 17.4 (Z
isomer), 14.0 (E isomer) and 13.6 (Z isomer). Analytical
(C.sub.21H.sub.28O.sub.2); calculated C=80.73 H=8.79; found C=80.60
H=8.77.
[0132] Representative procedure for preparation of
17-alkyl-2-methoxyestradiol analogs:
2-methoxy-17.beta.-methylestra-1,3,5(10)-triene-3-ol (Compound
#16): 17-Methylene-2-methoxyestra-1,3,5(10)triene-3-ol (471.9 mg,
1.58 mmol) was dissolved ethyl acetate (20 ml) in a Parr bottle
which was then flushed with Ar. 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.beta.-methylestra-1,3,5(10)-triene-3-ol. Selected
spectral data: .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 6.82 (s,
1H), 6.66 (s, 1H), 5.43 (s, 1H), 3.88 (s, 3H), 2.85-2.70 (m, 2H),
2.32-2.15 (m, 2H), 1.94-1.68 (m, 4H), 1.52-1.12 (m, 8H), 0.90 (d,
J=6.9 Hz, 3H), 0.61 (s, 3H). .sup.13C NMR (75 MHz, CDCl.sub.3)
.delta. 144.90, 143.75, 132.65, 130.11, 114.99, 108.51, 56.46,
55.21, 45.58, 44.85, 42.74, 39.39, 37.97, 30.65, 29.52, 28.38,
27.21, 24.83, 14.34, 12.44. Analytical (C.sub.20H.sub.28O.sub.2);
calculated C=79.96 H=9.39; found C=79.98 H=9.49.
[0133] Preparation of
3-sulfamate-2-methoxyestra-1,3,5(10)-trien-17-one (Compound #19):
Prepared as depicted in Scheme 3 using representative procedures.
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.06 (s, 1H), 6.93 (s,
1H), 4.93 (s, 2H), 3.88 (s, 3H), 2.86 (m, 2H), 2.58-1.26 (m, 313H),
0.92 (s, 1H).
[0134] Preparation of
3-sulfamate-2-ethoxyestra-1,3,5(10)-trien-17-one (Compound #20):
Prepared as depicted in Scheme 3 using representative procedures.
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.02 (s, 1H), 6.93 (s,
1H), 5.04 (br s, 2H), 4.12 (m, 2H), 3.73 (t, J=8.33 Hz, 1H), 2.78
(m, 2H), 2.28-1.17 (m, 13H), 1.43 (t, J=6.98 Hz, 3H), 0.78 (s,
3H).
[0135] Representative procedure for the preparation of triflic
esters: Preparation of
2-methoxy-3-trifluoromethanesulfonylestra-1,3,5(10)-trien-17-one
(Compound #22): 2-Methoxyestra-1,3,5(10)-trien-17-one-3-ol (2.4 g,
8.0 mmol) was dissolved in anhydrous dichloromethane (80 mL),
anhydrous pyridine (20 mL) was added and the mixture was cooled to
0.degree. C. Triflic anhydride (2 mL, 11.89 mmol, 1.5 eq) was added
drop wise, then allowed to warm to room temperature with stirring
for 18 h. The reaction mixture was poured into water (300 mL) and
additional dichloromethane (150 mL) was added. The layers were
separated, and the organic was washed with water (200 mL), 10% HCl
(2.times.100 mL) and brine (100 mL). The organic layer was dried
with MgSO.sub.4, filtered and solvent was removed under reduced
pressure. The product was purified using a SiO.sub.2 Biotage FLASH
apparatus using 5:1 hexanes ethyl acetate as eluent, obtain 2.3801
g (5.51 mmol, 68% yield) as final product. .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. 6.96 (s, 1H), 6.95 (s, 1H), 3.90 (s, 3H), 2.87
(dd, J=4.2, 6.4 Hz, 2H), 2.61-2.95 (m, 8H), 1.74-1.36 (m, 5H), 0.94
(s, 3H).
[0136] Synthesis of
2-methoxy-3-(vinyl)estra-1,3,5(10)-triene-17-one (Compound #25):
Tributyl(vinyl)tin(I) (200 .mu.L, 0.6 mmol), lithium chloride (85
mg, 2 mmol), dichlorobis(triphenylphosphine)palladium (II) (10 mg,
0.05 mmol), 2,6-di-tert-butyl-4-methylphenol (2 mg) was added to a
solution of
2-methoxy-3-trifluoromethanesulfoneestra-1,3,5(10)-triene-17-one
(216 mg, 0.5 mmol) in anhydrous DMF (4 mL) at room temperature. The
reaction was heated at 90.degree. C. for 18 h and then cooled to
room temperature. HF-pyridine (4 mL) was added and the reaction
mixture was stirred for an additional 6 h. The mixture was diluted
with EtOAc (20 mL) and filtered through Celite filter agent. The
combined organics were washed with 1 N HCl (1.times.100 mL), water
(1.times.100 mL), and brine (1.times.100 mL), dried with sodium
sulfate, filtered, and concentrated via rotary evaporation to give
a semi solid viscous oil. The product was purified on a flash
silica gel column eluted with a hexane/EtOAc 5:1 mixture to give a
white solid product (75 mg, 30%); .sup.1H NMR (CDCl.sub.3) .delta.
7.20 (s, 1H), 7.00 (dd, J=14, 11 Hz, vinyl, 1H), 6.82 (s, 1H), 5.70
(d, J=18, vinyl, 1H), 5.22 (d, J=11, vinyl, 1H) 3.88 (s, 3H), 2.85
(m, 2H), 2.52 (dd, J=3 Hz, 1H), 2.44-1.88 (m, 6H), 1.72-1.35 (m,
6H), 0.95 (s, 3H).
[0137] Alternate synthesis of
2-methoxy-3-(vinyl)estra-1,3,5(10)-triene-17.beta.-ol (Compound
#25a): Into a solution of
3-triflate-2-methoxyestra-1,3,5(10)-trien-17.beta.-ol (218 mg, 0.5
mmol, prepared as in Shi J. Am. Chem. Soc. 2002, 124, 6921) in 4 mL
DMF (anhydrous), was added tributyl(vinyl)Tin(I) (200 .mu.L, 0.6
mmol), lithium chloride (85 mg, 2 mmol),
dichlorobis(triphenylphosphine)palladium (II) (10 mg, 0.05 mmol),
2,6-di-tert-butyl-4-methylphenol (2 mg) at room temperature. The
reaction was heated at 90.degree. C. for 24 h and then cooled to
room temperature. After adding HF-pyridine (4 mL) the reaction
mixture was stirred for 16 h. After diluting with EtOAc (20 mL) the
reaction mixture was filtered through Celite filter agent and the
combined organics were washed with 1 N HCl (1.times.100 mL), water
(1.times.100 mL), and brine (1.times.100 mL), then dried with
sodium sulfate, filtered, and concentrated via rotary evaporation
to give a semi solid viscous oil. The product was purified on a
flash silica gel column eluted with a hexane/EtOAc 2:1 mixture to
give a white solid product (140 mg, 60%). .sup.1H NMR (CDCl.sub.3
.delta. 7.20 (s, 1H), 7.00 (dd, J=14, 11 Hz, vinyl, 1H) 6.80 (s,
1H), 5.70 (d, J=18 Hz, vinyl, 1H), 5.22 (d, J=11 Hz, vinyl, 1H),
3.85 (s, 3H), 2.72 (t, J=4 Hz, 1H), 2.85 (dd, J=5 Hz, 2H),
2.44-1.88 (m, 6H), 1.80-1.15 (m, 8H), 0.80 (s, 3H).
[0138] Synthesis of 2-methoxy-3-(carboxylic
acid)estra-1,3,5(10)-triene-17-one (Compound #28): Potassium
acetate (786 mg, 8 mmol), palladium (II) acetate (40 mg, 0.1 mmol),
1,1-bis(diphenylphosphine)-ferrocene (dppp) (200 mg, 0.4 mmol) were
added to a solution of #22 (175 mg, 0.4 mmol) in 4 mL DMSO
(anhydrous) at room temperature. The mixture was purged with CO gas
for five minutes at room temperature then heated at 50.degree. C.
for 18 h under CO (balloon) and cooled to room temperature. After
diluting with water, the reaction mixture was extracted with
CH.sub.2Cl.sub.2 (1.times.20 mL) washed with 1 N HCl (1.times.10
mL), water (1.times.10 mL), and brine (1.times.10 mL), then dried
with sodium sulfate, filtered, and concentrated via rotary
evaporation to give a dark viscous oil. The product was purified on
a flash silica gel column eluted with a hexane/EtOAc 1:2 mixture to
give a white solid product (40 mg, 30%). .sup.1H NMR (CDCl.sub.3)
.delta. 10.92 (br s, 1H), 7.91 (s, 1H), 6.95 (s, 1H), 4.05 (s, 3H),
2.85 (dd, J=5 hz, 2H), 2.42-1.85 (m, 6H), 1.65-1.15 (m, 8H), 0.80
(s, 3H).
[0139] Representative procedure for conversion of 3-(carboxyl
acid)estrane derivative to 3-(carboxamide)estrane analog: Synthesis
of 2-methoxy-3-carboxamideestra-1,3,5(10)-triene-17-one (Compound
#31): A 150 mg portion of #28 (0.5 mmol) was dissolved in
SOCl.sub.2 at room temperature and then reaction mixture was heated
at 50-60.degree. C. for 2 h until gas evolution ceased. The
remaining SOCl.sub.2 was removed under vacuum to give a dark
viscous oil. The product was diluted with THF anhydrous (5 mL) and
NH.sub.3 gas was bubbled for 5 minutes. Soon a white precipitate of
NH.sub.4Cl formed. The reaction mixture was diluted with THF,
filtered and evaporated to give viscous product. The product was
purified on a flash silica gel column eluted with a hexane/EtOAc
1:2 mixture to give a white solid product (60 g, 30%). .sup.1H NMR
(CDCl.sub.3) .delta. 7.95 (s, 1H), 7.82 (br s, 1H), 6.95 (s, 1H),
5.72 (br s, 1H), 3.95 (s, 3H), 2.95 (dd, J=5 Hz, 2H), 2.42-1.85 (m,
6H), 1.65-1.15 (m, 8H), 0.80 (s, 3H).
[0140] Representative preparation of 3-(benzophenone imine)estrane
derivatives--Preparation of
3-dibenzylimine-2-methoxyestra-1,3,5(10)-trien-17-one:
3-Trifluoromethanesulfonyl-2-methoxyestra-1,3,5(10)-trien-17-one
(908 mg, 2.367 mmol) was dissolved in anhydrous toluene (6 mL) and
Pd(OAc).sub.2 (30 mg, 5.7 mol %), rac BINAP (135 mg, 9.2 mol %),
Cs.sub.2CO.sub.3 (1.0729 g, 3.29 mmol, dried overnight in vacuum
oven) and benzophenone imine (436 L, 2.6 mmol) were added. The
mixture was refluxed for 60 h, cooled to room temperature, and
diethyl ether (200 mL) was then added. The resulting mixture was
filtered through celite, and solvent was removed under reduced
pressure. The mixture was purified using SiO.sub.2 with a Biotage
FLASH apparatus with 5:1 hexanes: ethyl acetate as eluent. Obtain
906 mg (1.96 mmol, 58% yield) as final product. .sup.1H NMR (300
MHz, CDCl.sub.3) .delta. 7.86-7.14 (m, 10H), 6.67 (s, 1H), 6.37 (s,
1H), 3.65 (s, 3H), 2.80-2.59 (m, 2H), 2.58-1.85 (m, 8H), 1.69-1.23
(m, 5H), 0.93 (s, 3H).
[0141] Representative procedure for hydrolysis of 3-(benzophenone
imine)estrane derivatives to 3-aminoestrane
derivatives--Preparation of
3-amino-2-methoxyestra-1,3,5(10)-trien-17-one (Compound #34):
3-Dibenzylimine-2-methoxyestra-1,3,5(10)-trien-17-one (906 mg, 1.96
mmol) was dissolved in THF (10 mL) and 2M HCl (2 mL) was added with
stirring for 4 h. The reaction mixture was partitioned between 2:1
hexanes:ethyl acetate and 0.5M HCl (100 mL each). The aqueous layer
was adjusted to pH=10 with 10M NaOH and then washed with
dichlormethane (2.times.150 mL). The organic layer was washed with
brine (100 mL), dried with Na.sub.2SO.sub.4, filtered and solvent
was removed under reduced pressure. Obtain 438 mg (1.46 mmol, 75%
yield) as final product. .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.
6.74 (s, 1H), 6.49 (s, 1H), 3.95 (br s, 2H), 3.84 (s, 3H),
2.89-2.69 (m, 2H), 2.60-1.88 (m, 8H), 1.74-1.34 (m, 5H), 0.93 (s,
3H).
[0142] 3-Amino-2-ethoxyestra-1,3,5(10)-trien-17-one (Compound #35):
Prepared as depicted in Scheme 3 using the representative
procedures: .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 6.74 (s, 1H),
6.50 (s, 1H), 4.05 (q, J=6.8 Hz, 2H), 3.70 (br s, 2H), 2.86-2.73
(m, 2H), 2.60-1.91 (m, 13H), 1.44 (t, J=6.8 Hz, 3H), 0.93 (s,
3H).
[0143] Representative procedure to prepare 3-fluoroestrane analogs:
Preparation of 3-fluoro-2-methoxyestra-1,3,5(110)-trien-17-one
(Compound #40): 3-Amino-2-methoxyestra-1,3,5(10)-trien-17-one (#34,
439 mg, 1.468 mmol) was dissolved in ethanol (10 mL) and HBF.sub.4
(48% aqueous, 5 mL) and cooled to 0.degree. C. NaNO.sub.2 (124 mg,
1.797 mmol in water (0.5 mL) was added and stirred for 2.5 h.
Diethyl ether (500 mL) was added, caused an oil to form. The
supernatant was decanted off, and the flask was dried under vacuo
causing the oil to foam. This foam was stored overnight under vacuo
at 80.degree. C. and was subsequently purified using SiO.sub.2
Biotage FLASH apparatus with 2:1 hexanes:ethyl acetate as eluent.
Obtain 92 mg (0.305 mmol, 21% yield). .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. 6.91 (d, J=8.3 Hz, 1H), 6.82 (d, J=12.1 Hz,
1H), 3.88 (s, 3H), 2.95-2.77 (m, 2H), 2.61-1.92 (m, 8H), 1.73-1.36
(m, 5H), 0.94 (s, 3H). This compound was reduced with NaBH.sub.4 at
room temperature or LiAlH.sub.4 at -78.degree. C. to give the
corresponding 17 alcohol.
[0144] Preparation of
3-Formamide-2-methoxyestra-1,3-5(10)-trien-17-ol (Compound #34):
Same reaction procedure as the preparation of #88, except the
17-ester was cleaved with methanolic NaOH. .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. 8.7 (d, J=11.7 Hz, rotamer 2), 8.44 (s, rotamer
1) and 8.10 (s, rotamer 2, total 1H), 7.72 (br s) and 7.58 (m,
exchangeable amide H), 6.90 (d, J=16.2 Hz, rotamer 1), 6.84 (s,
rotamer 1, 1H) (total of all H from 8.7-6.84 (m, 4H), 3.88 (s,
rotamer 1) and 3.86 (s, rotamer 2, total 3H), 3.76 (m, 1H),
2.88-2.77 (m, 2H), 2.40-1.13 (m, 14H), 0.81 (s, 3H).
[0145] Preparation of
3-formamide-2-methoxyestra-1,3,5(10)-triene-17-one (Compound 43a):
Prepared as depicted in Scheme 3 using representative procedures.
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 8.73 (d, J=11.5 Hz,
rotamer 1), 8.46 (d, J=1.9 Hz, rotamer 1) and 8.13 (s, rotamer 2,
total 1H), 7.76 (br s, rotamer 1) and 7.68-7.61 (m, rotamer 2,
total 1H), 6.94 (s, rotamer 1) and 6.85 (s, rotamer 2), 6.82 (s,
1H) (all H between 8.73 and 6.82 total 4H), 4.16-4.01 (m, 2H),
2.94-2.76 (m, 2H), 2.62-1.15 (m, 15H), 1.47 (app t, J=7.0 and 7.7
Hz, 3H), 0.94 (s, 3H).
[0146] Preparation of
3-Formamide-2-ethoxyestra-1,3-5(10)-trien-17-one (Compound #44a):
Prepared as depicted in Scheme 3 using representative procedure for
#88. .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 8.74 (d, J=11.7 Hz,
rotamer 2), 8.46 (d, J=2.08 Hz, rotamer 1) and 8.13 (s, rotamer 2,
total 1H), 7.76 (br s, rotamer 1) and 7.69-7.61 (m, rotamer 2,
total 1H), 6.88 (d, J=15 Hz, rotamer 1), 6.28 (s, 1H) (all protons
from 8.74-6.28 total 4H), 4.16-4.01 (m, 2H), 2.95-2.77 (m, 2H),
2.63-1.18 (m, 16H), 0.94 (s, 3H).
[0147] Preparation of 3-sulfamate-2-methoxyestra-1,3,5(10)-triene
(Compound #46): Prepared as depicted in Scheme 4 using
representative procedures. .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 7.06 (s, 1H), 6.97 (s, 1H), 4.96 (br s, 2H), 3.89 (s, 3H),
2.85-2.78 (m, 2H), 2.33-2.19 (m, 2H), 2.00-1.11 (m, 13H), 0.77 (s,
3H).
[0148] Preparation of 3-Triflic-2-methoxyestra-1,3,5(10)-triene
(Compound #49): Same general procedure as the preparation of #22.
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 6.97 (s, 1H), 6.92 (s,
1H), 3.89 (s, 3H), 2.93-2.75 (m, 2H), 2.35-2.19 (m, 2H), 2.03-1.06
(m, 15H), 0.77 (s, 3H).
[0149] Preparation of 3-Vinyl-2-methoxyestra-1,3,5(10)-triene
(Compound #52): Prepared as depicted in Scheme 4 using the
representative procedures: .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 7.20 (s, 1H), 7.00 (dd, J=14.5, 11.2 Hz, 1H), 6.85 (s, 1H),
5.71 (d, J=17.7 Hz, 1H), 5.22 (d, J=11.3 Hz, 1H), 3.85 (s, 3H),
2.88-2.78 (m, 2H), 2.35-2.22 (m, 2H), 2.01-1.10 (m, 11H), 0.95 (t,
J=7.54, 2H), 0.77 (s, 3H).
[0150] Preparation of
(2-Methoxyestra-1,3,5(10)-trien-3-yl)-3-carboxamide (Compound #58):
A 500 mL round-bottomed flask fitted with an overhead stirrer, a
Claisen adaptor with a thermocouple probe and carbon monoxide
inlet, and a vacuum inlet, was charged with
2-methoxyestra-1,3,5(10)-trien-3-yl-(trifluoromethyl)sulfonate
(#49, 24 g, 57.35 mmol), anhydrous dimethylformamide (185 mL),
palladium (II) chloride (0.500 g, 2.87 mmol),
1,3-bis(diphenylphospino)propane (2.37 g, 5.74 mmol), and
1,1,1,3,3,3-hexamethyldisilazane (48 mL, 229 mmol). The resulting
yellow solution was stirred and evacuated, flushed with carbon
monoxide (balloon) several times, then heated to 102.degree. C. for
12 h. Additional palladium (II) chloride (0.500 g),
1,3-bis(diphenylphospino)propane (2.40 g), and hexamethyldisilazane
(30 mL) were added and the mixture was re-evacuated, charged with
carbon monoxide, and heated at 102.degree. C. for an additional 12
h. Methanol (50 mL) was added and, after several minutes, the dark
solution was partitioned with ethyl acetate (1000 mL) and 2 N
sulfuric acid (1000 mL). The aqueous phase was extracted with ethyl
acetate (2.times.250 mL), the combined organic extracts were washed
with additional sulfuric acid (500 mL), and the aqueous phase was
back-extracted with ethyl acetate (2.times.250 mL), and the total
combined dark organic layers were washed with saturated aqueous
sodium bicarbonate (500 mL) and dried over sodium sulfate (400 g).
Suction-filtration through a plug of silica gel 60 (136 g) and
concentration afforded 19 g of crude product as a red paste. This
was purified by flash chromatography using 430 g of silica gel, and
eluting with 10% ethyl acetate-dichloromethane. The
product-containing fractions were concentrated, then taken up in
acetone and re-concentrated (2.times.2000 mL). The yellow solid was
then slurried in n-heptane (200 mL) overnight and isolated by
suction-filtration. Removal of residual solvent, and drying to a
constant weight over 3 h in a vacuum oven at 70.degree. C. and 0.5
torr, afforded 5.07 g (28% overall) of #58 as an off-white powder.
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 10.82 (br s, 1H), 7.99
(s, 1H), 6.95 (s, 1H), 4.05 (s, 3H), 2.95 (dd, 2H, J=5), 2.15 (m
2H,) 2.02-1.65 (m, 6H), 1.55-1.15 (m, 8H), 0.80 (s, 3H). C NMR (125
MHz, CDCl.sub.3) .delta. 167.6, 156.1, 147.1, 132.9, 130.0, 116.0,
108.7, 56.2, 54.0, 45.2, 41.2, 40.7, 39.0, 38.6, 28.8, 28.2, 26.7,
25.5, 20.8, 17.8.
[0151] Preparation of 3-amino-2-methoxyestra-1,3,5(10)-triene
(Compound #61): Prepared as depicted in Scheme 4 using
representative procedures. .sup.1H NMR, (CDCl.sub.3,) .delta. 6.80
(1H, s, aromatic), 6.48 (1H, s, aromatic), 3.87 (3H, s), 3.65 (2H,
broad, NH.sub.2), 2.75 (2H, dd, J=5.0, 3.0 Hz), 2.25 (2H, m), 1.97
(2H, m), 1.80-1.05 (11H, m), 0.80 (3H, s).
[0152] Preparation of 3-formamide-2-methoxyestra-1,3,5(10)-triene
(Compound #64): Prepared as depicted in Scheme 4 using
representative procedures. .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 8.70 (d, J=11.7 Hz, rotamer 2), 8.44 (d, J=1.7 Hz, rotamer
2) and 8.09 (s, rotamer 1, total 1H), 7.71 (br s, rotamer 1) and
7.62-7.53 (m, rotamer 2, total 1H), 6.90 (d, J=10.4 Hz, rotamer 1),
6.85 (s, 1H) (all protons from 8.70-6.85 total 4H), 3.88 (s,
rotamer 1) and 3.86 (s, rotamer 2, total 3H), 2.91-2.77 (m, 2H),
2.33-2.18 (m, 2H), 2.00-1.08 (m, 13H), 0.77 (s, 3H).
[0153] Representative procedure for preparation of sulfamate
derivatives:
17-methylene-3-sulfamate-2-methoxyestra-1,3,5(10)-triene (Compound
#70): Sodium hydride (268 mg, 11.2 mmol) was added to anhydrous DMF
(40 mL) and cooled to 0.degree. C., then
17-methylene-2-methoxyestra-1,3,5(10)-triene-3-ol in anhydrous DMF
(8 mL) was added drop wise. The mixture was stirred for 2 h at room
temperature. Sulfamoyl chloride (prepared freshly as in Peterson et
al J. Med. Chem. 1992, 35, 3991) was added in portions at 0.degree.
C. and stirred overnight at room temperature. The mixture was
poured into water (100 mL) and washed with ethyl acetate
(3.times.100 mL). The combined organic layers were washed with
water (4.times.100 mL) and brine (100 mL). The organics were dried
with MgSO.sub.4, filtered and solvent was removed under reduced
pressure to obtain a white solid. After drying under vacuo for 24
h, obtain 441 mg product (70% yield, 1.17 mmol). .sup.1H NMR (300
MHz, CDCl.sub.3) .delta. 7.07 (s, 1H), 6.98 (s, 1H), 4.96 (s, 2H),
4.71 (s, 2H), 3.91 (s, 3H), 2.93-2.73 (m, 2H), 2.65-1.15 (m, 13H),
0.85 (s, 3H).
[0154] Preparation of
3-triflic-17-methylene-2-methoxyestra-1,3,5(110)-triene (Compound
#73): Prepared as depicted in Scheme 5 using representative
procedures. .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 6.98 (s, 1H),
6.93 (s, 1H), 4.73-4.68 (m, 2H), 3.90 (s, 3H), 2.88-2.78 (m, 2H),
2.64-2.50 (m, 1H), 2.41-1.18 (m, 13H), 0.85 (s, 3H).
[0155] Alternate preparation of 3-carboxamide estrane derivatives
directly from triflate--Preparation of
2-Methoxy-17-methyleneestra-1,3,5(110)-triene-3-carboxamide
(Compound #82): General procedure based on Tetrahedron Letters,
1998, 39, 2835-2838. A 250 mL three-necked flask equipped with an
overhead stirrer, thermocouple, and nitrogen inlet, was charged
with 2-Methoxy-17-methyleneestra-1,3,5(10)-trien-3-yl
(trifluoromethyl)-sulfonate #73 (20.0 g, 46.5 mmol), palladium (II)
chloride (0.41 g, 2.3 mmol), 1,3-bis(diphenylphosphino) propane
(1.9 g, 4.6 mmol), 1,1,1,3,3,3-hexamethyldisilazane (38.8 mL, 186
mmol) and anhydrous dimethylformamide (150 mL). The resulting
orange solution was evacuated and back-filled with nitrogen three
times, then evacuated and back-filled with carbon monoxide three
times. The reaction was warmed to 100.degree. C. and stirred under
a carbon monoxide atmosphere (balloon) for 18 hours, during which
time the solution became dark red. The heat was removed, methanol
(40 mL) was added, and the solution allowed to stir for 10 minutes.
The solution was poured into ethyl acetate (1 L) and extracted with
2 N H.sub.2SO.sub.4 (1 L). The aqueous layer was extracted with
ethyl acetate (3.times.500 mL). Each of the ethyl acetate extracts
was washed with 2 N H.sub.2SO.sub.4 (500 mL). The combined organic
phases were washed with saturated aqueous sodium bicarbonate (1 L)
and dried over sodium sulfate (500 g). This suspension was
suction-filtered through a bed of silica gel 60 (102 g), and the
filtrate concentrated to dryness affording 16.02 g (106% recovery)
of crude #82. The crude product was purified on silica gel 60 (500
g, flash column) eluting with methylene chloride (2 L) then 2%
methanol-methylene chloride (4 L) then 4% methanol-methylene
chloride (4 L). Chromatography failed to remove all impurities so
fractions containing product were combined, concentrated to
dryness, and repurified on silica gel 60 (500 g, flash column)
eluting with methylene chloride (2 L) then 1:5 ethyl
acetate-methylene chloride. Concentration of the pure fractions
(TLC, 1:5 ethyl acetate-methylene chloride, R.sub.f=0.3, UV
detection) and drying in a vacuum oven at 80.degree. C., afforded
12.3 g (81% overall) of compound #82 as a light-yellow solid with
residual methylene chloride by NMR. The material was dissolved in
10:1 acetone/methanol (1.35 L), concentrated to dryness to remove
residual solvents, and dried in vacuo to yield 11.36 g of compound
#16 (75% overall yield) as a light-yellow solid. .sup.1H-NMR
(showed 0.09 wt % acetone present), .sup.13C-NMR and Mass Spectral
analysis were consistent with desired structure. .sup.1H NMR (300
MHz, CDCl.sub.3) .delta. 7.94 (s, 1H), 7.63 (br s, 1H), 6.95 (s,
1H), 5.70 (br s, 1H), 4.71 (s, 2H), 3.97 (s, 3H), 2.97-2.82 (m,
2H), 2.65-1.19 (m, 13H), 0.86 (s, 3H). .sup.13C NMR (CDCl.sub.3,
125 MHz) .delta. 167.2, 161.4, 155.8, 146.3, 132.6, 129.6, 118.0,
108.4, 100.9, 55.9, 53.5, 44.9, 44.2, 38.2, 35.6, 29.4, 28.4, 27.4,
26.4, 23.9, 18.5.
[0156] Preparation of
3-amino-17-methylene-2-methoxyestra-1,3,5(110)-triene (Compound
#85): Prepared as depicted in Scheme 5 using representative
procedures. .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 6.78 (s, 1H),
6.49 (s, 1H), 4.72-4.67 (m, 2H), 3.85 (s, 2H), 3.67 (br s, 2H),
2.83-2.71 (m, 2H), 2.63-2.48 (m, 1H), 2.41-1.14 (m, 13H), 0.85 (s,
3H).
[0157] Preparation of
3-amino-17-methylene-2-ethoxyestra-1,3,5(10)-triene (Compound #86):
Prepared as depicted in Scheme 5 using representative procedures.
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 6.77 (s, 1H), 6.48 (s,
1H), 4.69 (s, 2H), 4.06 (q, J=7.0 Hz, 2H), 3.70 (br s, 2H),
2.87-2.65 (m, 2H), 2.63-2.48 (m, 1H), 2.40-2.11 (m, 2H), 2.03-1.17
(m, 10H), 1.43 (t, J=6.8 Hz, 3H), 0.84 (s, 3H).
[0158] Representative preparation of 3-formamide estrane
derivatives: Preparation of
3-formamide-17-methylene-2-methoxyestra-1,3,5(10)-triene (Compound
#88): 3-Amino-17-methylene-2-methoxyestra-1,3,5(10)-triene (293 mg,
0.99 mmol) was dissolved in chloroform (1 mL) and trimethylacetyl
formic anhydride (170 mg, 1.30 mmol, prepared as in Vlietstra et al
Recueil, 1982, 101, 460) was added. The mixture was stirred 40 min,
after which, the solvent was removed under reduced pressure. The
product was purified by titurating with hexanes and isolating the
precipitate by decanting after the precipitate was centrifuged.
Obtain 150 mg white powder (0.417 mmol, 42% yield). .sup.1H NMR
(300 MHz, CD.sub.3OD) .delta. 8.27 (s, 1H), 7.88 (s, 1H), 6.94 (s,
1H), 4.72-4.67 (m, 2H), 3.88 (s, rotamer 1) and 3.86 (s, rotamer 2,
total 3H), 2.87-2.76 (m, 2H), 2.66-1.19 (m, 13H), 0.87 (s, 3H).
[0159] Preparation of
3-formamide-17-methylene-2-ethoxyestra-1,3,5(110)-triene (Compound
#89): Prepared as depicted in Scheme 5 using representative
procedures. .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 8.73 (d,
J=11.7 Hz, rotamer 2), 8.46 (d, J=1.88 Hz, rotamer 2) and 8.11 (s,
total 1H), 7.75 (br s, rotamer 1) and 7.62 (br s, rotamer 2,
exchangeable amide H), 6.90 (d, J=18 Hz, rotamer 1), 6.84 (s, all
protons from 8.73-6.84 total 4H), 4.70 (s, 2H), 4.16-4.02 (m, 2H),
2.93-2.79 (m, 2H), 2.66-1.16 (m, 16H), 0.85 (s, 3H).
[0160] Preparation of
3-sulfamate-17-ethylene-2-methoxyestra-1,3,5(110)-triene (Compound
#94): Prepared as depicted in Scheme 6 using representative
procedures. .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.06 (s, 1H),
6.96 (s, 1H), 5.24-5.13 (m, 1H), 4.96 (s, 2H), 3.90 (s, 3H),
2.90-2.78 (m, 2H), 2.51-1.20 (m, 16H), 0.94 (s, Z isomer) and 0.80
(s, E isomer, total 3H, ratio 5:1 respectively).
[0161] Preparation of
3-amino-17-ethylene-2-methoxyestra-1,3,5(10)-triene (Compound
#109): Prepared as depicted in Scheme 6 using representative
procedures. .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 6.76 (s, 1H),
6.48 (s, 1H), 5.22-5.12 (m, 1H), 3.85 (s, 3H), 3.66 (br s, 2H),
2.86-2.66 (m, 2H), 2.53-2.16 (m, 4H), 2.00-1.20 (m, 12H), 0.93 (s,
Z isomer) and 0.80 (s, E isomer, total 3H, ratio 5:1
respectively).
[0162] Preparation of
3-formamide-17-ethylene-2-methoxyestra-1,3,5(10)-triene (Compound
#112): Prepared as depicted in Scheme 6 using representative
procedures. .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 8.70 (d,
J=11.7 Hz, rotamer 2), 8.44 (d, J=1.89 Hz, rotamer 1) and 8.09 (s,
rotamer 2, total 1H), 7.72 (br s, rotamer 1) and 7.63-7.52 (m,
rotamer 2, exchangable amide H), 6.90 (d, J=14.7 Hz, rotamer 1),
6.84 (s, 1H) (all protons from 8.70-6.84 total 4H), 5.24-5.14 (m,
1H), 3.89 (s, rotamer 1) and 3.86 (s, rotamer 2, total 3H),
2.90-2.78 (m, 2H), 2.53-2.18 (m, 5H), 2.03-1.23 (m, 11H), 0.93 (s,
Z isomer) and 0.80 (s, E isomer, total 3H, ratio 5:1
respectively).
[0163] Preparation of
3-sulfamate-17-methyl-2-methoxyestra-1,3,5(10)-triene (Compound
#118): Prepared as depicted in Scheme 7 using representative
procedures. .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.06 (s, 1H),
6.97 (s, 1H), 4.95 (s, 2H), 3.90 (s, 3H), 2.89-2.74 (m, 2H),
2.35-2.15 (m, 2H), 1.99-1.12 (m, 12H), 0.91 (d, J=6.8 Hz, 3H), 0.62
(s, 3H).
[0164] Preparation of
3-sulfamate-17-methyl-2-ethoxyestra-1,3,5(10)-triene (Compound
#119): Prepared as depicted in Scheme 7 using representative
procedures. .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.042 (s,
1H), 6.96 (s, 1H), 4.99 (s, 2H), 4.21-4.06 (m, 2H), 2.87-2.74 (m,
2H), 2.32-1.12 (m, 17H), 0.90 (d, J=5.30 Hz, P isomer) and 0.79 (d,
J=3.77 Hz, a isomer, total 3H, ratio 4:1 respectively), 0.61 (s,
3H).
[0165] Preparation of
3-amino-17-methyl-2-methoxyestra-1,3,5(10)-triene (Compound #133):
Prepared as depicted in Scheme 7 using representative procedures.
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 6.77 (s, 1H), 6.48 (s,
1H), 3.85 (s, 3H), 2.87-2.66 (m, 2H), 2.33-2.14 (m, 2H), 1.95-1.11
(m, 14H), 0.89 (d, J=6.78 Hz, P isomer) and 0.79 (d, J=3.77 Hz, a
isomer, total 3H, ratio 4:1 respectively), 0.61 (s, 3H).
[0166] Preparation of
3-formamide-17-methyl-2-methoxyestra-1,3,5(10)-triene (Compound
#136): Prepared as depicted in Scheme 7 using representative
procedures. .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 8.70 (d,
J=11.7 Hz, rotamer 2), 8.43 (d, J=1.89 Hz, rotamer 1) and 8.09 (s,
rotamer 2, total 1H), 7.72 (br s) and 7.61-7.52 (m, exchangeable
H), 6.90 (d, J=10.9 Hz, rotamer 1), 6.85 (s, 1H) (all protons from
8.70-6.85 total 4H), 3.88 (s, rotamer 1) and 3.86(s, rotamer 2,
total 3H), 2.93-2.73 (.mu., 2H), 2.34-1.10 (.mu., 14H), 0.90 (d,
J=6.78 Hz, .beta. isomer) and 0.79 (d, J=3.7 Hz, .alpha. isomer,
total 3H, ratio 4:1 respectively), 0.61 (s, 3H).
EXAMPLE 2
[0167] Determination of in vitro anti-proliferative activity of
substituted estradiol analogs: In vitro anti-proliferative or
anti-mitogenic activity was determined using a commercially
available cell-based assay in 96-well tissue culture plates with
assessment of proliferation by evaluating DNA synthesis through
incorporation into DNA of immuno-reactive (BrdU) nucleotides. The
cell types used are commercially available (MDA-MB-231: breast
cancer; U87-MG: glioblastoma; PC3: prostate cancer; HUVEC:
non-transformed early passage human umbilical vein endothelial
cells). These assays, and the many assay variations possible to
determine in vitro anti-proliferative or anti-mitogenic activity,
are well known to those skilled in the art. The concentration which
causes 50% inhibition of proliferation (IC.sub.50) was estimated
from a does-response curve generally carried out with a range of
concentrations from .gtoreq.100 microg/mL to .ltoreq.0.01
microg/mL. The results of the tests are shown below in Table IV.
TABLE-US-00004 TABLE IV IC.sub.50 for IC.sub.50 for IC.sub.50 for
IC.sub.50 for Compound # MDA-MB-231 (.mu.M) U87-MG (.mu.M) PC3
(.mu.M) HUVEC (.mu.M) 1 0.69 .+-. 0.14 1.48 .+-. 0.62 1.08 .+-.
0.50 0.68 .+-. 0.15 2 0.650 .+-. 0.23 0.14 .+-. 0.07 3 3.99 .+-.
1.12 2.68 .+-. 0.22 2.22 .+-. 0.13 0.52 .+-. 0.14 4 11.7 10.5 13.7
12.6 7 2.20 .+-. 0.52 7.35 .+-. 0.43 8.49 .+-. 2.49 1.32 .+-. 0.56
10 0.58 .+-. 0.31 0.26 .+-. 0.23 13 2.63 .+-. 0.20 2.37 .+-. 0.34
1.15 .+-. 0.40 0.37 .+-. 0.08 16 3.33 .+-. 0.23 9.39 .+-. 0.43 6.46
.+-. 2.31 2.40 .+-. 0.45 19 0.60 .+-. 0.43 0.80 .+-. 0.23 0.51 .+-.
0.24 0.18 .+-. 0.04 20 7.52 .+-. 2.04 19.82 .+-. 2.32 8.93 .+-.
0.61 2.59 .+-. 0.25 25 2.57 .+-. 0.29 2.20 .+-. 0.06 1.43 .+-. 0.40
0.58 .+-. 0.01 25a 8.93 .+-. 0.61 7.81 .+-. 1.30 6.34 .+-. 0.08
1.96 .+-. 0.05 28 >100 .+-. 0 >100 .+-. 0 >100 .+-. 0
>100 .+-. 0 31 0.7 .+-. 0.12 1.07 .+-. 0.15 0.7 .+-. 0.03 0.79
.+-. 0.20 34 46.91 .+-. 8.15 81.54 .+-. 20.37 >100 .sup. 23.42
.+-. 4.14 35 10.64 .+-. 2.98 26.05 .+-. 1.40 9.25 .+-. 1.58 16.46
.+-. 2.02 37 2.48 .+-. 0.50 10.83 .+-. 1.04 9.79 .+-. 1.93 2.32
.+-. 0.38 40 2.94 .+-. 0.80 25.98 .+-. 3.38 9.26 .+-. 0.73 2.10
.+-. 0.23 43 0.78 .+-. 0.01 2.70 .+-. 0.22 1.34 .+-. 0.02 0.07 .+-.
0.02 .sup. 43A 1.22 .+-. 0.60 0.94 .+-. 0.09 0.61 .+-. 0.27 0.53
.+-. 0.22 .sup. 44A 24.87 .+-. 5.63 21.10 27.70 .+-. 0.62 13.17
.+-. 2.20 46 0.38 .+-. 0.15 0.72 .+-. 0.04 0.38 .+-. 0.21 0.21 .+-.
0.15 52 1.86 .+-. 0.22 3.14 .+-. 0.52 2.12 .+-. 0.64 0.58 .+-. 0.04
58 5.23 .+-. 1.95 4.52 .+-. 1.3 2.58 .+-. 0.36 1.42 .+-. 0.41 61
7.62 .+-. 0.43 10.20 .+-. 3.78 8.63 .+-. 0.18 6.43 .+-. 1.32 64
8.29 .+-. 1.64 23.24 .+-. 2.0 9.24 .+-. 0.59 5.80 .+-. 0.73 70 0.56
.+-. 0.02 0.70 .+-. 0.06 0.91 .+-. 0.32 0.58 .+-. 0.03 73 >100
.sup. >100 .sup. >100 .sup. >100 .sup. 82 0.19 .+-. 0.08
0.25 .+-. 0.02 0.25 .+-. 0.01 0.12 .+-. 0.06 85 2.11 .+-. 0.57 5.51
.+-. 0.46 2.40 .+-. 0.39 1.00 .+-. 0.19 86 2.13 .+-. 0.12 7.29 .+-.
2.26 2.75 .+-. 0.22 2.31 .+-. 0.05 88 0.30 .+-. 0.08 0.53 .+-. 0.24
0.24 .+-. 0.06 0.22 .+-. 0.03 89 2.41 .+-. 0.13 7.73 1.59 .+-. 0.19
2.10 .+-. 0.02 94 0.58 .+-. 0.02 0.64 .+-. 0.09 0.66 .+-. 0.05 0.59
.+-. 0.00 109 1.94 .+-. 0.27 2.35 .+-. 0.41 0.92 .+-. 0.11 0.72
.+-. 0.10 112 0.56 .+-. 0.02 0.77 .+-. 0.06 0.74 .+-. 0.01 0.58
.+-. 0.00 113 11.98 19.13 20.64 7.08 118 0.66 .+-. 0.10 0.64 .+-.
0.01 0.82 .+-. 0.06 0.59 .+-. 0.1 119 1.55 .+-. 0.39 6.41 0.83 0.91
.+-. 0.09 133 7.31 .+-. 0.58 14.87 .+-. 0.01 7.16 .+-. 1.42 2.65
.+-. 0.77 136 0.61 .+-. 0.00 0.64 .+-. 0.01 0.70 .+-. 0.10 0.59
.+-. 0.00
[0168] 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.
* * * * *