U.S. patent application number 10/624765 was filed with the patent office on 2004-06-17 for therapeutic applications of estrogenic carboxylic acids.
This patent application is currently assigned to Southern Illinois University. Invention is credited to Adler, Stuart R., Banz, William J., Dandliker, Walter B., Hou, Yuqing, Meyers, Cal Y., Winters, Todd A..
Application Number | 20040116398 10/624765 |
Document ID | / |
Family ID | 22222383 |
Filed Date | 2004-06-17 |
United States Patent
Application |
20040116398 |
Kind Code |
A1 |
Meyers, Cal Y. ; et
al. |
June 17, 2004 |
Therapeutic applications of estrogenic carboxylic acids
Abstract
Provided are methods employing estrogenic compounds for:
repressing weight gain or reducing weight in male patients;
treating or preventing prostate cancer and peri- or post-menopausal
symptoms; treating estrogen-responsive conditions that no longer
respond to treatment with conventional steroidal estrogens;
treating or preventing estrogen-responsive uterine cancer, breast
cancer, and ovarian follicle atresia; inducing ovulation to
increase fertility; oral contraception; treating or preventing
diseases or conditions caused or prolonged by free radicals;
treating or preventing cardiovascular disease, hyperlipidemia or
hypercholesterolemia, and hyperglycemia; improving body fat
distribution; and treating or preventing Alzheimer's disease,
osteoporosis, and pattern baldness. Also provided are methods for
treating or preventing prostatic diseases including benign prostate
hyperplasia and other related conditions, androgen-responsive
pathological conditions in males, and methods for male birth
control and chemical castration, employing estrogenic carboxylic
acids.
Inventors: |
Meyers, Cal Y.; (Carbondale,
IL) ; Banz, William J.; (Carterville, IL) ;
Adler, Stuart R.; (Creve Coeur, MO) ; Winters, Todd
A.; (Murphysboro, IL) ; Hou, Yuqing;
(Carbondale, IL) ; Dandliker, Walter B.; (La
Jolla, CA) |
Correspondence
Address: |
SENNIGER POWERS LEAVITT AND ROEDEL
ONE METROPOLITAN SQUARE
16TH FLOOR
ST LOUIS
MO
63102
US
|
Assignee: |
Southern Illinois
University
|
Family ID: |
22222383 |
Appl. No.: |
10/624765 |
Filed: |
July 22, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10624765 |
Jul 22, 2003 |
|
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|
09338823 |
Jun 23, 1999 |
|
|
|
6608111 |
|
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60090344 |
Jun 23, 1998 |
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Current U.S.
Class: |
514/182 |
Current CPC
Class: |
A61K 31/19 20130101 |
Class at
Publication: |
514/182 |
International
Class: |
A61K 031/56 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 22, 1999 |
WO |
PCT/US99/13940 |
Claims
What is claimed is:
1. A method for repressing weight gain or reducing weight in a male
patient, comprising administering (+)-Z-bisdehydrodoisynolic acid
in a dosage effective to repress weight gain or reduce weight to a
male patient suffering from, or disposed to, weight gain.
2. The method of claim 1, wherein said dosage is in the range of
from about 0.1 .mu.g/kg/day to about 100 mg/kg/day.
3. A method for treating or preventing a disease, condition, or
symptom selected from the group consisting of prostatic disease,
peri- or post-menopausal symptoms, an estrogen-responsive condition
that no longer responds to treatment with conventional steroidal
estrogens, an estrogen-responsive uterine cancer, breast cancer,
ovarian follicle atresia, a disease or condition caused or
prolonged by free radicals, cardiovascular disease, hyperlipidemia,
hypercholesterolemia, hyperglycemia, Alzheimer's disease and
pattern baldness, comprising administering an estrogenic carboxylic
acid in a dosage effective to treat or prevent said disease,
symptom, or condition to a patient suffering from, or disposed to,
said disease, symptom, or condition.
4. The method of claim 3, wherein said estrogenic carboxylic acid
is selected from the group consisting of a doisynolic acid, an
allenolic acid, a phenylcyclohexenecarboxylic acid, a
hydroxyphenylcyclohexenecarbo- xylic acid, a
phenylcyclohexanecarboxylic acid, a hydroxyphenylcyclohexane-
carboxylic acid, a hydroxytetrahydroanthracenecarboxylic acid, and
a tetrahyroanthracenecarboxylic acid.
5. The method of claim 4, wherein said estrogenic carboxylic acid
is selected from the group consisting of (+)-doisynolic acid,
(-)-Z-bisdehydro-doisynolic acid, (+)-Z-bisdehydrodoisynolic acid,
(.+-.)-Z-bisdehydrodoisynolic acid, (-)-allenolic acid,
(+)-allenolic acid,
1-(p-hydroxyphenyl)-6-ethyl-5-methylcyclohexene-4-carboxylic acid,
1-(p-hydroxyphenyl)-2-ethyl-3-methylcyclohexene-4-carboxylic acid,
1-(p-hydroxyphenyl)-2-ethyl-3,5,5-trimethylcyclohexene-4-carboxylic
acid, 4-(p-hydroxyphenyl)-2,2,6,6-tetramethylcyclohexanecarboxylic
acid,
1-ethyl-6-hydroxy-2-methyl-1,2,3,4-tetrahydroanthracene-2-carboxylic
acid, 1-phenyl-2-ethyl-3-methylcyclohexene-4-carboxylic acid, and
1-phenyl-5,6-dimethylcyclohexene-4-carboxylic acid, or a
pharmaceutically acceptable salt, ester, or anhydride thereof.
6. The method of claim 5, wherein said estrogenic carboxylic acid
is selected from the group consisting of (-)-Z-bisdehydrodoisynolic
acid, (+)-Z-bisdehydrodoisynolic acid, and
(.+-.)-Z-bisdehydrodoisynolic acid.
7. The method of claim 3, wherein said dosage is in the range of
from about 0.1 .mu.g/kg/day to about 100 mg/kg/day.
8. A method for treating or preventing osteoporosis comprising
administering an estrogenic carboxylic acid selected from the group
consisting of a doisynolic acid, a phenylcyclohexenecarboxylic
acid, a hydroxyphenylcyclohexenecarboxylic acid, a
phenylcyclohexanecarboxylic acid, a
hydroxyphenylcyclohexanecarboxylic acid, a
hydroxytetrahydroanthracenecarboxylic acid, and a
tetrahydroanthracenecar- boxylic acid in a dosage effective to
treat or prevent osteoporosis to a male or female patient suffering
from, or disposed to, osteoporosis.
9. The method of claim 8 wherein said estrogenic carboxylic acid is
selected from the group consisting of (+)-doisynolic acid,
(-)-Z-bisdehydro-doisynolic acid, (+)-Z-bisdehydrodoisynolic acid,
(.+-.)-Z-bisdehydrodoisynolic acid,
1-(p-hydroxyphenyl)-6-ethyl-5-methylc- yclohexene-4-carboxylic
acid, 1-(p-hydroxyphenyl)-2-ethyl-3-methylcyclohex-
ene-4-carboxylic acid,
1-(p-hydroxyphenyl)-2-ethyl-3,5,5-trimethylcyclohex-
ene-4-carboxylic acid,
4-(p-hydroxyphenyl)-2,2,6,6-tetramethylcyclohexanec- arboxylic
acid, 1-ethyl-6-hydroxy-2-methyl-1,2,3,4-tetrahydroanthracene-2--
carboxylic acid, 1-phenyl-2-ethyl-3-methylcyclohexene-4-carboxylic
acid, and 1-phenyl-5,6-dimethylcyclohexene-4-carboxylic acid, or a
pharmaceutically acceptable salt, ester, or anhydride thereof.
10. The method of claim 9, wherein said estrogenic carboxylic acid
is selected from the group consisting of (-)-Z-bisdehydrodoisynolic
acid, (+)-Z-bisdehydrodoisynolic acid, and
(.+-.)-Z-bisdehydrodoisynolic acid.
11. The method of claim 8, wherein said dosage is in the range of
from about 0.1 .mu.g/kg/day to about 100 mg/kg/day.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a division of U.S. patent application
Ser. No. 09/338,823, filed Jun. 23, 1999, which claims priority
from U.S. Provisional Patent Application Serial No. 60/090,344,
filed Jun. 23, 1998.
FIELD OF THE INVENTION
[0002] The present invention relates to the field of pharmaceutical
therapeutics. More specifically, the present invention relates to
the use of estrogenic carboxylic acids in improved therapies for
the treatment of a variety of symptoms and disease conditions in
mammals. The present invention also relates to the field of
chemical synthesis, more specifically, the synthesis of estrogenic
carboxylic acids.
BACKGROUND OF THE INVENTION
[0003] A. Estrogens
[0004] Estrogens, such as (+)-17.beta.-estradiol (E2), have
physiological effects on males as well as females. In addition to
their activity in reproductive tissue, they promote rapid weight
gain in specific species, and have been marketed to fatten
livestock quickly. Trenkle, AH: "The Mechanisms of Action of
Estrogens in Feeds on Mammalian and Avian Growth." Proceedings of a
Symposium: The Use of Drugs in Animal Feed. National Academy of
Science, Washington D.C. 150-164 (1968); Meyers, U.S. Pat. No.
5,420,161. Estrogens have long been prescribed for their beneficial
effects by reducing susceptibility to osteoporosis and ameliorating
menopausal and postmenopausal symptoms. Evans S F, Davie M W: "Low
and Conventional Dose Transdermnal Oestradiol Are Equally Effective
at Preventing Bone Loss In Spine and Femur at All Post-Menopausal
Ages." Clin Endocrinol. 44:79-84 (1996); Agarwal S K, Judd H L:
"Menopause." Curr Ther Endocrinol Metab. 6:624-631 (1997).
Long-term clinical studies suggest that estrogens may be beneficial
in promoting cardiovascular health. Wilson P W: "The Impact of
Estrogen on Cardiovascular Disease." Perspective Studies: The
Framingham Study. Postgrad Med 51-53:89-90 (1989). More recently,
estrogens have shown promise as an adjunct in treatment of
Alzheimer's disease. Filley C M: "AlZheimer's Disease in Women." Am
J Obstet Gynecol 176:1-7 (1997). Unfortunately, some estrogenic
compounds administered in therapeutic doses are suspected
carcinogens in target tissues including breast and uterus. Persson
I: "Cancer Risk in Women Receiving Estrogen-Progestin Replacement
Therapy." Maturitas 23:S37-45 (1996).
[0005] Non-steroidal estrogens and antiestrogens, including
pharmaceuticals, environmental compounds, and phytochemicals, are
currently receiving significant attention. This is understandable
from the myriad potential applications increasingly being reported
for estrogenic compounds, e.g., treating menopause- and
post-menopause-related problems, as anti-carcinogens, alleviating
osteoporosis, for contraceptive use, in estrogen-replacement
therapy, treating prostatic disease, improving serum lipid
profiles, etc. The multiplicity of estrogenic effects now being
discovered has led many investigators to target specific
populations for treatment with estrogen agonists and antagonists.
Synthetic nonsteroidal compounds such as triphenylethylene
derivatives (e.g., tamoxifen), dihydronapthalene derivatives (e.g.,
nafoxidine), and benzothiophene derivatives (e.g., raloxifene)
exhibit estrogenic and anti-estrogenic activity in various tissues,
these respective compounds showing specific advantages in the
management of bone, uterine, serum cholesterol, and adipose tissue.
See, generally, Trenkle, AH: "The Mechanisms of Action of Estrogens
in Feeds on Mammalian and Avian Growth." Proceedings of a
Symposium: The Use of Drugs in Animal Feed. National Academy of
Science, Washington D.C. 150-164 (1968); Evans S F, Davie M W: "Low
and Conventional Dose Transdermal Oestradiol Are Equally Effective
at Preventing Bone Loss In Spine and Femur at All Post-Menopausal
Ages." Clin Endocrinol. 44:79-84 (1996); Agarwal S K, Judd H L:
"Menopause." Curr Ther Endocrinol Metab. 6:624-631 (1997); Wilson P
W: "The Impact of Estrogen on Cardiovascular Disease." Perspective
Studies:
[0006] The Framingham Study. Postgrad Med 51-53:89-90 (1989);
Filley C M: "AlZheimer's Disease in Women." Am J Obstet Gynecol
176:1-7 (1997); Persson I: "Cancer Risk in Women Receiving
Estrogen-Progestin Replacement Therapy." Maturitas 23:S37-45
(1996); Heer J, Billeter J R, Miescher K: "Totalsynthese der
racemischen bisdehydro-doisynolsure. ber oestrogene carbosuren IV."
Helv. Chim. Acta 28:1342-1354 (1945); Ke H Z, Chen H A, Simmons H
A, Qi H, Crawford D T, Pirie C M, Chidsey-Frink K L, Ma Y F, Jee W
S S, Thompson D D: "Comparative Effects of Droloxifene, Tamoxifen,
and Estrogen on Bone, Serum Cholesterol, and Uterine Histology in
the Ovariectomized Rat Model." Bone 20:31-39 (1997); Sato M, Rippy
M K, Bryant H U: "Raloxifene, Tamoxifen, Nafoxidine, or Estrogen
Effects on Reproductive and Nonreproductive Tissues in
Ovariectomized Rats." FASEB J 10:905-912 (1996); Dodge J A,
Glasebrook A L, Magee D A, Phillips D L, Sato M, Short L L, Bryant
H U: "Environmental Estrogens: Effects on Cholesterol Lowering and
Bone in the Ovariectomized Rat." J Steroid Biochem Molec Biol
59:155-161 (1996); Hart J E: "Endocrine Pathology of Estrogens:
Species Differences." Pharmac Ther 47:203-218 (1990); Heywood R,
Wadsworth P F: "The Experimental Toxicology of Estrogens." Pharmac
Ther 8:125-142 (1980); Baker V L, Draper M, Paul S, Allerheiligen
S, Glant M, Shifren J, Jaffe R B: "Reproductive Endocrine and
Endometrial Effects of Raloxifene Hydrochloride, A Selective
Estrogen Receptor Modulator, in Women with Regular Menstrual
Cycles." J Clin Endocrin Metab 83:6-13 (1998); DanZo B J:
"Environmental Xenobiotics May Disrupt Normal Endocrine Function by
Interfering with the Binding of Physiological Ligands to Steroid
Receptors and Binding Proteins." Environ Health Perspect
105:294-301 (1997); Baker V L, Jaffe R B: "Clinical Uses of
Antiestrogens." Obstet Gynecol Surv 51:45-59 (1996); Knight D C,
Eden J A: "A Review of the Clinical Effects of Phytoestrogens."
Obstet Gynecol 87:897-904 (1996); Cooper R L, Kavlock R J:
"Endocrine Disruptors and Reproductive Development: A
Weight-of-Evidence Overview." J Endocrinol 152:159-166 (1997);
Reubinoff B E, Wurtman J, Rojansky N, Adler D, Stein P, Schenker J
G, BrZeZinski A: "Effects of Hormone Replacement Therapy on Weight,
Body Composition, Fat Distribution, and Food Intake in Early
Postmenopausal Women: A Prospective Study." Fertil Steril
64:963-968 (1995).
[0007] B. Doisynolic Acids and Related Estrogenic Compounds
[0008] Doisynolic acids, named after their discoverer, Edward
Doisy, are estrogenic compounds originally obtained from alkali
fusion of estrone and equilenin. "Doisynolic acid," from estrone,
contains a phenolic moiety; and "bisdehydrodoisynolic acid" (BDDA),
from equilenin, possesses a .beta.naphtholic moiety. Both types are
seco-steroids, i.e., the steroidal D-ring is cleaved. See Miescher
K: "On Doisynolic Acids, A New Class of Estrogens." Chem Rev
43:367-384 (1948); Fieser L F, Fieser M: Natural Products Related
to Phenanthrene, 347-353 (3rd Ed., Reinhold Publishing Corp., New
York, N.Y. 1949). Meyers and Kolb reported the conversions of E2
and estrone under very mild conditions into doisynolic acids,
which, in turn, exhibited estrogenic and antiestrogenic activity
depending on dosage. Meyers C Y, Kolb V M: "Facile and Selective
Chlorination and Cleavage of Some Cyclanones and Cyclanols With the
CCl.sub.4-KOH-t-BuOH Reagent. In situ Conversions of Estrones and
Estradiols into Dichlorodoisynolic Acids." J Org Chem 43:1985-1990
(1978). A number of related pseudo-seco-steroid acids (most of them
containing only two rings or a shifted C ring) also have been
prepared. These compounds have been cited as exhibiting varying
degrees of estrogenicity. Meyers C Y, Kolb V M, Gass G H, Rao B R,
Roos C F, Dandliker W B: "Doisynolic-Type Acids--Uterotropically
Potent Estrogens which Compete Poorly with Estradiol for Cytosolic
Estradiol Receptors. J Steroid Biochem 31:393-404 (1988).
[0009] It has been reported that (.+-.)-Z-doisynolic acid is more
estrogenic than (+)-E-doisynolic acid (C-14, S configuration)
derived from estrone or E2. Anner G, Miescher K: Hydrierungs-Und
Umlagerungs-Reaktion in der Doisynolsure--Reihe. Oestrogene
Carbonsuren XII. Helv. Chim. Acta 29 (1946) 1889-1895; and Die
totalsyntheses von racemischen doisynolsuren XXI. ber oestrogene
carbonsueren. ibid 30:1422-1432 (1947). Of the Z and E isomers of
the doisynolic-type compounds, (.+-.)-Z-bisdehydrodoisynolic acid
[(.+-.)-Z-BDDA] has been reported to be among the most estrogenic
in vivo, rivaling or even surpassing estradiol for vaginal
cornification and uterotropism in the in vivo assays that were used
to determine the comparative estrogenicity. Miescher K: "On
Doisynolic Acids, A New Class of Estrogens." Chem Rev 43:367-384
(1948); Fieser L F, Fieser M: Natural Products Related to
Phenanthrene, 347-353 (3rd Ed., Reinhold Publishing Corp., New
York, N.Y. 1949); Meyers C Y, Kolb V M, Gass G H, Rao B R, Roos C
F, Dandliker W B: "Doisynolic-Type Acids--Uterotropically Potent
Estrogens which Compete Poorly with Estradiol for Cytosolic
Estradiol Receptors. J Steroid Biochem 31:393-404 (1988); Tschopp
E: "Wirksamkeit, organconzentration und ausscheidung der
7-methyl-bisdehydro-doisynolsure." Helv Physiol Pharmacol Acta
4:401-410 (1946); Tschopp E: "Die oestrogene wirkung der
bisdehydrodoisynolsure und ihre derivate." Helv Physiol Pharmacol
Acta 4:271-284 (1946); Rometsch R, Miescher K: "Die spaltung des
racemates der n-bisdehydro-doisynolsure. ber ostrogene carbonsuren
X." Helv Chim Acta 29:1231-1235 (1946); and Terenius L:
"Differential Inhibition In Vitro of 17.beta.-Estradiol Binding in
the Mouse Uterus and Vagina by Optical Antipodes of Estrogen."
Molec Pharmac 4:301-310 (1968). Additional assays of (.+-.)-Z-BDDA
for estrogenicity, based on the estrogen-dependent cell
proliferation in MCF-7 human mammary cancer cell line in culture,
have confirmed the high estrogenic potency of this compound. Meyers
C Y, Kolb V M, Gass G H, Rao B R, Roos C F, Dandliker W B:
"Doisynolic-Type Acids--Uterotropically Potent Estrogens which
Compete Poorly with Estradiol for Cytosolic Estradiol Receptors. J
Steroid Biochem 31:393-404 (1988); and Soto A M, Meyers C Y,
Sonnenschein C: "How Many Rings Can be Cleaved from a Steroidal
Estrogen While Preserving its Estrogenic Activity?" The Endocrine
Society, 70th Annual Meeting, Abstract (1988). And despite this
estrogenic potency, the (.+-.)-Z-BDDA has been reported to elicit
neither toxicity nor carcinogenicity, even at 1000-times the
estrogenic dosage. Meyers C Y, Kolb V M, Gass G H, Rao B R, Roos C
F, Dandliker W B: "Doisynolic-Type Acids--Uterotropically Potent
Estrogens which Compete Poorly with Estradiol for Cytosolic
Estradiol Receptors. J Steroid Biochem 31:393-404 (1988). It has
been reported that the (-) enantiomer of Z-BDDA is the one
responsible for the observed estrogenic potency. Anner G, Miescher
K: Hydrierungs--Und Umlagerungs-Reaktion in der
Doisynolsure--Reihe. Oestrogene Carbonsuren XII. Helv. Chim. Acta
29 (1946) 1889-1895; Die totalsyntheses von racemischen
doisynolsuren XXI. ber oestrogene carbonsueren. ibid 30:1422-1432
(1947); Tschopp E: "Wirksamkeit, organconzentration und
ausscheidung der 7-methyl-bisdehydro-doisynolsure." Helv Physiol
Pharmacol Acta 4:401-410 (1946); Tschopp E: "Die oestrogene wirkung
der bisdehydrodoisynolsure und ihre derivate." Helv Physiol
Pharmacol Acta 4:271-284 (1946); Rometsch R, Miescher K: "Die
spaltung des racemates der n-bisdehydro-doisynolsure. ber ostrogene
carbonsuren X." Helv Chim Acta 29:1231-1235 (1946); and Terenius L:
"Differential Inhibition In Vitro of 17.beta.-Estradiol Binding in
the Mouse Uterus and Vagina by Optical Antipodes of Estrogen."
Molec Pharmac 4:301-310 (1968).
[0010] One of the distinctive properties of estrogenic doisynolic
acids is their very low binding affinity to cytosolic estrogen
receptors when considered in context with their very high in vivo
activity. This anomaly was discovered by competitive binding
inhibition studies with .sup.3H-estradiol using estrogen receptors
extracted from rabbit uteri. Meyers C Y, Kolb V M, Gass G H, Rao B
R, Roos C F, Dandliker W B: "Doisynolic-Type Acids--Uterotropically
Potent Estrogens which Compete Poorly with Estradiol for Cytosolic
Estradiol Receptors. J Steroid Biochem 31:393-404 (1988). Unlabeled
estradiol has been reported to inhibit this binding strongly, while
the doisynoic acids have been reported to do so only about 1% as
well, despite being more active as estrogens in experimental
animals. More recent direct binding studies with ER .alpha. and ER
.beta. confirmed these results. Segaloff A.: "The Metabolism of
Estrogens with Particular Emphasis on Clinical Aspects of
Physiology and Function of Ovarian Hormones." Recent Progress in
Hormone Research 1949; IV:85-1 11; and Meyers C Y, Lutfi H G, Adler
S: "Transcriptional Regulation of Estrogen-Responsive Genes by
Non-Steroidal Estrogens: Doisynolic and Allenolic acids." J Steroid
Biochem Molec Biol 62:477-489 (1997).
[0011] Many recent studies have focused particularly on the in vivo
activity of (.+-.)-Z-bisdehydrodoisynolic acid, the most active and
easily available doisynolic acid. Competitive binding-inhibition
studies with uterine cytosolic estrogen receptors (ER) showed that
the binding affinity of (.+-.)-Z-BDDA was on the order of 0.01-0.03
of that of E2. Meyers C Y, Kolb V M, Gass G H, Rao B R, Roos C F,
Dandliker W B: "Doisynolic-Type Acids--Uterotropically Potent
Estrogens which Compete Poorly with Estradiol for Cytosolic
Estradiol Receptors. J Steroid Biochem 31:393-404 (1988); Soto A M,
Meyers C Y, Sonnenschein C: "How Many Rings Can be Cleaved from a
Steroidal Estrogen While Preserving its Estrogenic Activity?" The
Endocrine Society, 70th Annual Meeting, Abstract (1988). Recent
direct in vitro ER binding studies with human ER alpha (ER .alpha.)
and ER beta (ER .beta.) confirmed these results, in accord with the
binding affinities of (-)-Z-BDDA determined with mouse uterine ER
preparations in competitive binding-inhibition studies. Terenius L:
"Differential Inhibition In Vitro of 17.beta.-Estradiol Binding in
the Mouse Uterus and Vagina by Optical Antipodes of Estrogen."
Molec Pharmac 4:301-310 (1968); Segaloff A.: "The Metabolism of
Estrogens with Particular Emphasis on Clinical Aspects of
Physiology and Function of Ovarian Hormones." Recent Progress in
Hormone Research IV:85-111 (1949); and Meyers C Y, Lutfi H G, Adler
S: "Transcriptional Regulation of Estrogen-Responsive Genes by
Non-Steroidal Estrogens: Doisynolic and Allenolic acids." J Steroid
Biochem Molec Biol 62:477-489 (1997). Unlike most other estrogenic
compounds studied with these techniques, the BDDA compounds exhibit
a low binding affinity accompanied by a disproportionately high
biological activity. Without being bound by any particular theory,
it is believed that the classic estrogen receptor, ER, may not be
the exclusive receptor or pathway involved in mediating the actions
of Z-BDDA and other estrogenic compounds; and/or that doisynolic
acid compounds may act in vivo by some mechanism other than by
binding to estrogen cytosolic receptors to which estradiol,
estrone, etc., normally bind. See Meyers C Y, Kolb V M, Gass G H,
Rao B R, Roos C F, Dandliker W B: "Doisynolic-Type
Acids--Uterotropically Potent Estrogens which Compete Poorly with
Estradiol for Cytosolic Estradiol Receptors. J Steroid Biochem
31:393-404 (1988).
[0012] Differences in the activity of E2 and (.+-.)-Z-BDDA based on
other indices of estrogenic activity have also been observed.
Specifically, while the rate of weight gain of female mice
receiving E2 (e.g., 5 g/animal/day) was increased over that of the
control group, the rate of weight gain of female mice receiving
varying doses of (.+-.)-Z-BDDA (e.g, 5, 50, and 500 g/animal/day)
was actually diminished. Meyers, U.S. Pat. No. 5,420,161.
[0013] While estradiol and its 3-methyl ether have been reported to
be estrogenic in animals and humans, the 3-methyl ether of
(.+-.)-Z-BDDA has only been reported to be estrogenic in some
animals (but inactive in humans), and exhibits very little effect
on proliferating human MCF-7 cell growth. Soto A M, Meyers C Y,
Sonnenschein C: "How Many Rings Can be Cleaved from a Steroidal
Estrogen While Preserving its Estrogenic Activity?" The Endocrine
Society, 70th Annual Meeting, Abstract (1988). It has been
hypothesized that the enzyme or receptor responsible for the
conversion of the 3-methyl ether of estradiol to the estrogenic
phenolic estradiol is present in animals (including humans), while
that required for the similar conversion of the 3-methyl ether of
(.+-.)-Z-BDDA is present in some animals, but not humans.
[0014] Despite the above-discussed advances, there still exists a
need in the art for compounds exhibiting estrogen-like activity,
but lacking the undesirable side effects often observed in
connection with the use of conventional estrogens, for use in
methods for treating a wide variety of symptoms, conditions, and
diseases responsive to estrogens commonly employed at present.
[0015] C. Synthesis of Estrogenic Carboxylic Acids
[0016] In 1947 and 1948, Courrier, Horeau and Jacques (Courrier,
R.; Horeau, A.; Jacques, J. Sur un nouvel oestrogene artificial de
grande activit. Compt. rend. Soc. de biol. 1948, 141, 159-161;
Horeau, A.; Jacques, J. Structure moleculaire et activit
oestrogene: acides hydroxy-naphtylpropioniques substitutes. Acad.
Sc. 1947, 224, 862-864; Courrier, R.; Horeau, A.; Jacques, J.
L'acide allenolique et ses drivs. Acad. Sc. 1947, 224,1401-1407;
Courrier, R.; Horeau, A.; Jacques, J. Action de l'acide
dimethyl-ethyl-allenolique chez la femelle de cobaze qui allaite.
Compt. rend. Soc. de biol. 1947, 141, 747; Jacques, J.; Horeau, A.
Structure moleculaire et activit oestrogene (VI). Prparation de
quelques drivs de l'acide amphihydroxynaphtyl .beta.-propionique
(acide allenolique). Bull. Soc. Chim. France, 1948, 711-716)
reported the syntheses and biological studies of a series of
estrogenic compounds derived from
3-[2-(6-hydroxynaphthyl)]propionic acid 1, which was named
allenolic acid in honor of Dr. E. Allen. Of these compounds,
(-)-3-[2-(6-methoxynaphthyl)]-2,2-dimethyl-pentanoic acid 2 was
found to exhibit the strongest estrogenic activity in animals,
including rats, cats, chicks, and guinea pigs, while the (+)
enantiomer 3 showed only one-fifth the estrogenicity of 2
(Terenius, L. Inhibition of 17.beta.-estradiol uptake on mouse
uterus by doisynolic acid and allenolic acid derivatives: an in
vitro differentiation between true oestrogens and pro-oestrogens.
Acta Pharmacol. et Toxicol., 1967, 25, 313-322; Herbai, G.
Separation of Growth Inhibition Potency from Oestrogenicity in
Different Weak Oestrogenic Drugs of Various Chemical Structures,
Acta Endocrinologica, 1971, 68, 249-263). Later, the (-)
enantiomer, 2, was marketed by G. D. Searle & Company under the
trade name Vallestril.RTM. for the treatment of postmenopausal
symptoms (Crawley, G. C. Hormones-nonsteroidal estrogens. In
Kirk-Othmer Encyclo. Chem. Technol. 3rd Ed; Grayson, Martin,
Eckroth, David, Eds; Wiley: New York, 1980; vol. 12, 670-671).
[0017] Although 2 was highly estrogenic in animals, equivalent to
17.beta.-estradiol (E2), it was not found to have the same effects
in women as E2. In clinical trials, high dosages were required to
elicit strong estrogenic responses from women (Sturnick, M. I.;
Gargill, S. L. Clinical assay of a new synthetic estrogen:
Vallestril. New England J Med., 1952, 247, 830-834; Schneeberg, N.
G.; Perczek, L.; Nodine, J. H.; Perloff, W. H. Methallenstril, a
new synthetic estrogen. J Am. Med. Assoc. 1956, 161, 1062-1067),
and thus 2 was eventually removed from the market. 1
[0018] In 1967, Terenius (Terenius, L. Inhibition of
17.beta.-estradiol uptake on mouse uterus by doisynolic acid and
allenolic acid derivatives: an in vitro differentiation between
true oestrogens and pro-oestrogens. Acta Pharmacol. et Toxicol.,
1967, 25, 313-322) proposed that 2 was a pro-estrogen, and that the
true estrogen is its free phenolic form, i.e., compound 4, based on
a study of the inhibition of 17.beta.-estradiol uptake in mouse
uterus by those compounds. In 1971, Herbai (Herbai, G. Separation
of Growth Inhibition Potency from Oestrogenicity in Different Weak
Oestrogenic Drugs of Various Chemical Structures, Acta
Endocrinologica, 1971, 68, 249-263) reported that in mice, compound
4 exhibited a 100-fold stronger activity with regard to both
inhibition of weight gain and sulfate incorporation than compound
2. However, the (+) enantiomer of 4, compound 5, caused significant
depression of sulfate incorporation without the corresponding
effects on weight gain. Some years later, Soto et al. (Soto, A. M.;
Meyers, C. Y.; Sonnenschein, C. How Many Rings Can Be Cleaved from
a Steroidal Estrogen while Preserving its Estrogenic Activity? The
Endocrine Society, 70th Annual Meeting, Abstract (1988)) found that
while 2 showed very little effect in human MCF-7 cell
proliferation, its phenolic form, 4, was found to be highly
effective, suggesting that the low estrogenicity of 2 in women is
due to human inability to cleave the methyl group from the ethereal
oxygen. 2
[0019] Currently, there is a great deal of research interest in
selective estrogen receptor modulators (SERMs) (Baker, V. L.;
Draper, M.; Paul, S.; Allerheiligen, S.; Glant, M.; Shifren, J.;
Jaffe, R. B. Reproductive endocrine and endometrial effects of
raloxifene hydrochloride, a selective estrogen receptor modulator,
in women with regular menstrual cycles. J. Clin. Endocrinol.
Metab., 1998, 83, 6-13). SERMs have many potential medical
applications, such as in treating postmenopausal symptoms,
preventing osteoporosis, and hormonal therapy for prostate cancer,
while eliminating the unwanted side effects. For example,
raloxifene is marketed by Eli Lilly under the trade name
Evista.RTM. to prevent osteoporosis in postmenpausal women while
having little effect on other reproductive organs. Recent studies
on the physiological effects of (+)- and
(-)-cis-bisdehydrodoisynolic acids (cis-BDDA) in rats indicated
that these compounds could be used in a number of therapeutic
applications (Banz, W. J.; Winters, T. A.; Hou, Y.; Adler, S.;
Meyers, C. Y. Comparative Effects of (-)-, (+)- and
(.+-.)-Z-Bisdehydrodoisynolic Acids and Estradiol on Body Weight,
Food Intake and Metabolic Parameters in Male and Female Rats.
Hormone and Metabolic Research, 1998, 30, 730-736). More
importantly, (+)- and (-)-cis-BDDA have different physiological
effects on various organs in intact rats. As estrogenic carboxylic
acids, 4 and 5 have been shown to have similar in vitro and in vivo
biological properties to cis-BDDA (Meyers, C. Y.; Lutfi, H. G.;
Adler, S. Transcriptional regulation of estrogen-responsive genes
by non-steroidal estrogens: Doisynolic and allenolic acids. J.
Steroid Biochem. Molec. Biol., 62, 477-489 (1997)).
[0020] 3-[2-(6-methoxynaphthyl)]-2,2-dimethylpentanoic acid has an
asymmetric center on the benzylic carbon. Thus, there exist two
enantiomers, as indicated by structures 2 and 3, above. Previous
syntheses all yielded a racemic mixture of 2 and 3. Thus, a
resolution process was required to obtain the desired enantiomer,
as in the case of Vallestril.RTM.. Jacques and Horeau reported that
quinine could be used to resolve the two enantiomers by forming two
diastereomeric salts (Jacques, J.; Horeau, A. Structure molculaire
et activite oestrogne (VII). Ddoublement optique de l'acide
.alpha.,.alpha.-dimthyl .beta.-thyl allenolique. iBull. Soc. Chim.
France, 1949, 301-303).
[0021] Jacques and Horeau first reported the synthesis of
(.+-.)-3-[2-(6-methoxynaphthyl)]-2,2-dimethylpentanoic acid in 1947
and obtained a patent in 1949 (Scheme 1) (Jacques, J.; Horeau, A.
Structure moleculaire et activit oestrogene (VD). Prparation de
quelques drivs de l'acide amphihydroxynaphtyl .beta.-propionique
(acide allenolique). Bull. Soc. Chim. France, 1948, 711-716;
Jacques, J.; Horeau, A. Naphthalene derivatives having estrogenic
acitivity. Fr. Pat. 941,289 (1949)). 3
[0022] In 1948, Wieland and Miescher (Wieland, P.; Miescher, K.
Estrogenic carboxylic acids. XXVI. Derivatives of alkylated
.beta.-naphthylvaleric acids. Helv. Chim. Acta, 1948, 31,
1844-1854) reported a different synthesis of a racemic mixture of 2
and 3, and Gay and Horeau (Gay, R.; Horeau, A. Molecular structure
and estrogenic activity. XV. Preparation of
2,2-dialkyl-3-(6-methoxy-2-naphthyl)pentanoic acids and
2,2-dialkyl-3-(6-methoxy-2-naphthyl)hexanoic acids (derivatives of
allenolic acid). Bull. Soc. Chim. France, 1955, 955-962) also
synthesized a racemic mixture through a similar route (Scheme 2).
These syntheses (Schemes 1 and 2) are multistep processes. After
each step, separation of the intermediate product must be performed
before it is used for the next reaction. Thus, additional
chemicals, energy, and manpower are needed, which increases the
cost of production and lowers the overall yield of the desired
product. 4
[0023] Ciba Ltd. patented the shortest reported synthesis of
racemic mixture of 2 and 3 so far in literature (Scheme 3) (Ciba
Ltd, Naphthalenepropionic Acid, Swiss Patent 261,123 (1949); Ciba
Ltd, Naphthalenepropionic Acids and Derivatives thereof, British
Patent 652,003 (1951)). Although there is only one step in this
process, the yield of the product was not high due to self-coupling
reactions. 5
[0024] All of these syntheses lead to a racemic mixture containing
equal amounts of 2 and 3. Due to the different biological
properties of enantiomers 2 and 3, a resolution step must be
performed to separate and isolate each enantiomer for
pharmaceutical use, which also significantly increases the cost of
production. In addition, the undesired enantiomer (50% of the
racemic mixture) generated in the resolution process may be wasted
if it is not used in other applications.
[0025] In the absence of a commercial source of 4 and 5, a one-pot,
asymmetric synthesis of either 4 or 5 is needed in the art.
SUMMARY OF THE INVENTION
[0026] The present invention provides methods of using estrogenic
carboxylic acids and other non-steroidal estrogen-like compounds to
treat or prevent a variety of conditions and diseases now being
treated with conventional estrogens such as estradiol, ethinyl
estradiol, estrone, or Premarin. The methods disclosed herein are
based in part on the emerging realization that the female hormones
produced in males, and conversely male hormones produced in
females, have far reaching effects in health and disease, affording
new approaches to a variety of therapies. Further, the use of the
estrogenic compounds disclosed herein in the methods described
below should result in improved therapies lacking the undesirable
side effects often seen in connection with the use of conventional
estrogens.
[0027] Thus, in one aspect, the present invention provides a method
for repressing weight gain or reducing weight in a male patient,
comprising administering (+)-Z-bisdehydrodoisynolic acid in a
dosage effective to repress weight gain or reduce weight to a male
patient suffering from, or disposed to, weight gain.
[0028] In another aspect, the present invention provides a method
for treating or preventing prostate cancer, comprising
administering an estrogenic carboxylic acid in a dosage effective
to treat or prevent prostate cancer to a patient suffering from, or
disposed to, prostate cancer. The estrogenic carboxylic acid can
also be used to maintain prostate cancer patients who have been
previously treated with inhibitors of gondadotropin releasing
hormone (GnRH) secretion or of testosterone. The predominant
hormonal treatment now in use for prostate cancer consists of
monthly injections of leuprolide, an antagonist of GnRH. Hot
flashes resulting from this treatment are a common complaint. In
addition, leuprolide, a polypeptide, may give rise to an immune
response on continued use. In contrast, the estrogenic carboxylic
acids of the present invention are almost certainly
non-immunogenic. These compounds should reduce the size of the
testes, thereby ameliorating the effects of prostate hyperplasia,
limiting the spread of prostate cancer cells.
[0029] In another aspect, the present invention provides a method
for treating or preventing peri- or post-menopausal symptoms,
comprising administering an estrogenic carboxylic acid in a dosage
effective to treat or prevent peri- or post-menopausal symptoms to
a patient suffering from, or disposed to, said menopausal symptoms.
The present estrogenic carboxylic acids can be used in place of
conventional estrogens in hormone replacement therapy in
menopause.
[0030] In another aspect, the present invention provides a method
for treating an estrogen-responsive condition that no longer
responds to treatment with conventional steroidal estrogens,
comprising administering an estrogenic carboxylic acid in a dosage
effective to repress, reduce, or otherwise ameliorate said
condition to a patient suffering from said condition.
[0031] In yet another aspect, the present invention provides a
method for treating or preventing an estrogen-responsive uterine
cancer, comprising administering an estrogenic carboxylic acid in a
dosage effective to treat or prevent said cancer to a patient
suffering from, or disposed to, said cancer.
[0032] In yet another aspect, the present invention provides a
method for treating or preventing breast cancer, comprising
administering an estrogenic carboxylic acid in a dosage effective
to treat or prevent said cancer to a patient suffering from, or
disposed to, breast cancer.
[0033] These methods of treating uterine cancer and breast cancer
are based on the estrogenic, antiestrogenic, and antioxidant
properties of the present estrogenic carboxylic acids.
[0034] In another aspect, the present invention provides a method
for treating or preventing ovarian follicle atresia, comprising
administering an estrogenic carboxylic acid in a dosage effective
to treat or prevent ovarian follicle atresia to a patient suffering
from, or disposed to, atresia.
[0035] In a further aspect, the present invention provides a method
for inducing ovulation to increase fertility, comprising
administering an estrogenic carboxylic acid in a dosage effective
to induce ovulation to a patient suffering from, or disposed to,
ovulatory disorder. The estrogenic carboxylic acid can be
administered during the mid-portion of the first part of the
menstrual cycle, for example, for five days, starting at the fifth
day of said menstrual cycle.
[0036] In yet a further aspect, the present invention provides a
method for oral contraception, comprising administering an
estrogenic carboxylic acid in a dosage effective to prevent
ovulation to said patient throughout the menstrual cycle, starting
at day one thereof and continuing throughout said menstrual cycle
to about day 21. This method is especially useful for treatment of
a patient not suitable for treatment with a steroidal estrogen, for
example one who is a tobacco smoker, an obese patient, a patient
suffering from breast disease, or a patient prone to producing
emboli. In obese patients, this method provides the added benefit
of promoting concomitant weight loss. In these methods, the
estrogenic carboxylic acid can be administered in combination with
a progestin.
[0037] In another aspect, the present invention provides a method
for treating or preventing a disease or condition caused or
prolonged by free radicals, comprising administering an estrogenic
carboxylic acid in a dosage effective to treat or prevent said
disease or condition to a patient suffering from, or disposed to,
said disease or condition.
[0038] Another aspect of the present invention provides a method
for treating or preventing cardiovascular disease, comprising
administering an estrogenic carboxylic acid in a dosage effective
to treat or prevent cardiovascular disease to a patient suffering
from, or disposed to, cardiovascular disease.
[0039] In another aspect, the present invention provides a method
for treating or preventing hyperlipidemia or hypercholesterolemia,
comprising administering an estrogenic carboxylic acid in a dosage
effective to treat or prevent hyperlipidemia or
hypercholesterolemia to a patient suffering from, or disposed to,
hyperlipidemia or hypercholesterolemia.
[0040] In another aspect, the present invention provides a method
for treating or preventing hyperglycemia, comprising administering
an estrogenic carboxylic acid in a dosage effective to treat or
prevent hyperglycemia to a patient suffering from, or disposed to,
hyperglycemia.
[0041] Yet another aspect of the present invention involves a
method for improving body fat distribution, comprising
administering an estrogenic carboxylic acid in a dosage effective
to improve body fat distribution to a patient suffering from, or
disposed to, abnormal body fat distribution.
[0042] A further aspect of the present invention relates to a
method for treating or preventing Alzheimer's disease, comprising
administering an estrogenic carboxylic acid in a dosage effective
to treat or prevent Alzheimer's disease to a patient suffering
from, or disposed to, Alzheimer's disease.
[0043] Yet a further aspect of the present invention relates to a
method for treating or preventing osteoporosis, comprising
administering an estrogenic carboxylic acid in a dosage effective
to treat or prevent osteoporosis to a patient suffering from, or
disposed to, osteoporosis.
[0044] In still another aspect, the present invention provides a
method for treating or preventing pattern baldness, comprising
administering an estrogenic carboxylic acid in a dosage effective
to treat or prevent pattern baldness to a patient suffering from,
or disposed to, pattern baldness. Such patients include both males
and females. In balding men, hair growth should be stimulated by
the reduction of testosterone levels produced by feedback
inhibition of the pituitary occasioned by the rise in estrogen.
[0045] In another aspect, the present invention provides a method
for treating or preventing a prostatic disease or condition,
comprising administering an estrogenic carboxylic acid in a dosage
effective to treat or prevent a prostatic disease or condition to a
patient suffering from, or disposed to, such disease or condition.
(+)-Z-BDDA, (-)-Z-BDDA, (-)-HAA, (+)-HAA, can be used in this
method, with (+)-Z-BDDA being preferred. Examples of prostatic
diseases and conditions amenable to such treatment include, but are
not limited to, prostate cancer, benign prostate hypertrophy, and
prostatitis. These and other prostatic diseases and conditions can
be treated without negative side effects such as testis shrinkage,
inhibition of spermatogenesis, gynecomastia, or other feminizing
effects in males in accordance with this method.
[0046] In another aspect, the present invention provides a method
for treating or preventing an androgen-responsive pathological
condition in a male, comprising administering an estrogenic
carboxylic acid in a dosage effective to treat or prevent said
pathological condition to a male patient suffering from, or
disposed to, said pathological condition.
[0047] In yet another aspect, the present invention provides a
method of birth control, comprising administering an estrogenic
carboxylic acid in a dosage effective to inhibit spermatogenesis in
a male. Compounds useful in this method include, but are not
limited to, (-)-Z-BDDA, (-)-HAA, and (+)-HAA.
[0048] In a still further aspect, the present invention provides a
method for chemical castration in a male, comprising administering
an estrogenic carboxylic acid in a dosage effective to shrink the
testis or cause a loss of libido and/or impotence in a male.
Compounds useful in this method include, but are not limited to,
(-)-Z-BDDA, (-)-HAA, and (+)-HAA.
[0049] Treatment of the foregoing symptoms, conditions, and
diseases with the compounds of the present invention should be
accompanied by fewer side effects than are often observed in
connection with the use of conventional estrogens.
[0050] In any of the foregoing methods, the estrogenic therapeutic
compound most preferably is an estrogenic carboxylic acid, such as,
for example, a doisynolic acid, an allenolic acid, a
phenylcyclohexenecarboxy- lic acid, a
hydroxyphenylcyclo-hexenecarboxylic acid, a
phenylcyclohexanecarboxylic acid, a
hydroxyphenylcyclohexanecarboxylic acid, a
hydroxytetrahydro-anthracenecarboxylic acid, or a
tetrahyroanthracene-carboxylic acid. More specifically, the
estrogenic carboxylic acid can be, for example, (+)-doisynolic
acid, (-)-Z-bisdehydrodoisynolic acid, (+)-Z-bisdehydrodoisynolic
acid, (.+-.)-Z-bisdehydro-doisynolic acid (Z-BDDA), (-)-allenolic
acid, (+)-allenolic acid,
1-(p-hydroxyphenyl)-6-ethyl-5-methylcyclohexene-4-car- boxylic
acid, 1-(p-hydroxyphenyl)-2-ethyl-3-methylcyclohexene-4-carboxylic
acid,
1-(p-hydroxyphenyl)-2-ethyl-3,5,5-trimethylcyclohexene-4-carboxylic
acid, 4-(p-hydroxyphenyl)-2,2,6,6-tetramethylcyclohexanecarboxylic
acid,
1-ethyl-6-hydroxy-2-methyl-1,2,3,4-tetrahydroanthracene-2-carboxylic
acid, 1-phen yl-2-ethyl-3-methylcyclohexene-4-carboxylic acid, and
1-phenyl-5,6-dimethylcyclohexene-4-carboxylic acid. Derivatives of
such compounds (e.g., a pharmaceutically acceptable salt, ester, or
anhydride) may also be used. In the methods disclosed herein, these
estrogenic carboxylic acids can be used alone or in
combination.
[0051] In yet another aspect, the present invention provides a
direct one-pot synthesis to produce esters of
3-[2-(6-methoxynaphthyl)]-2,2-dime- thylpentanoic acid from
commercially available starting material. These esters can then be
easily hydrolyzed under basic or acidic conditions to yield the
corresponding acids 2 or 3, discussed above.
[0052] Further scope of the applicability of the present invention
will become apparent from the detailed description and drawings
provided below. However, it should be understood that the following
detailed description and examples, while indicating preferred
embodiments of the invention, are given by way of illustration only
since various changes and modifications within the spirit and scope
of the invention will become apparent to those skilled in the art
from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0053] The above and other objects, features, and advantages of the
present invention will be better understood from the following
detailed description taken in conjunction with the accompanying
drawings, all of which are given by way of illustration only, and
are not limitative of the present invention, in which:
[0054] FIG. 1 shows results of Experiment 2 of Example 2, i.e., the
effects of(-)-, (+)-, and (.+-.)-Z-bisdehydrodoisynolic acid
(Z-BDDA) and (+)-17.beta.b-estradiol on uterus weight in rats on
treatment for 5-6 weeks [.sup.1significantly different from vehicle
(p<0.05) and .sup.2significantly different from (-)-Z-BDDA
(p<0.05), n=5/treatment, all values are the mean.+-.SEM].
[0055] FIG. 2 shows results of Experiment 1 of Example 2, i.e., the
effects of (+)- and (.+-.)-Z-bisdehydrodoisynolic acid (Z-BDDA) and
(+)-17.beta.estradiol on percent weight change in male and female
rats on treatment for 4 weeks [.sup.1significantly different from
control (p<0.05); .sup.2significantly different from vehicle
(p<0.05); .sup.3 significantly different from estradiol
(p<0.05); and .sup.4Significantly different from (.+-.)-Z-BDDA
(p<0.05), n=5/treatment. All values are the mean .+-.SEM].
[0056] FIG. 3 shows results of Experiment 2 of Example 2, i.e., the
effects of(-)-, (+)-, and (.+-.)-Z-bisdehydrodoisynolic acid
(Z-BDDA) and (+)-17.beta.estradiol on % weight change in male and
female rats on treatment for 5-6 weeks [.sup.1significantly
different from vehicle (p<0.05) and .sup.2significantly
different from estradiol (p<0.05), n=5/treatment, all values are
the mean.+-.SEM].
[0057] FIGS. 4a-4f show the effects of (-)- and
(+)-Z-bisdehydrodoisynolic acids (BDDA), (-)- and
(+)-hydroxyallenolic acid (HAA), and (+)-17.beta.-estradiol (E) on
prostate histology in male rats on treatment for 6 weeks (Example
4). Photomicrographs represent hemotoxylin and eosin stains of
paraffin sections photographed at 20.times.. Representative panels
were treated as labeled at 0.1 .mu.g/g bodyweight.
[0058] FIGS. 5a-5f show the effects of (-)- and
(+)-Z-bisdehydrodoisynolic acids (BDDA), (-)- and
(+)-hydroxyallenolic acid (HAA), and (+)-17.beta.-estradiol (E) on
testis histology in male rats on treatment for 6 weeks (Example 4).
Photomicrographs represent hemotoxylin and eosin stains of paraffin
sections photographed at 20.times.. Representative panels were
treated as labeled at 0.1 .mu.g/g bodyweight.
[0059] FIG. 6 shows the results of the lag time oxidation studies
described in Example 5. Compounds shown on the x-axis, from left to
right: daidzein, genistein, 4-hydroxy-tamoxifen, (+)-allenolic
acid, (-)-allenolic acid, (+)-Z-bisdehydrodoisynolic acid,
(-)-Z-bisdehydrodoisynolic acid. Each compound was tested at a
concentration of 10.sup.-4, 10.sup.-5, 10.sup.-6, and 10.sup.-7 M.
The results obtained at each of these concentrations is shown for
each compound, from left to right as a vertical bar,
respectively.
DETAILED DESCRIPTION OF THE INVENTION
[0060] Although the following detailed description is provided to
aid those skilled in the art in practicing the present invention,
it should not be construed to unduly limit the present invention.
Modifications and variations in the embodiments discussed herein
can be made by those of ordinary skill in the art without departing
from the spirit or scope of the present inventive discovery.
[0061] The present methods utilize the unique properties of
doisynolic acid and related estrogenic compounds (especially
related estrogenic carboxylic acids) in animal (particularly human)
therapy and research. Based on the properties of these compounds,
together with present methods of treating prostate, breast, and
uterine cancer, osteoporosis, atresia, Alzheimer's disease, etc.,
improved therapies are proposed for these disorders. These
improvements stem from the unique properties of the estrogenic
therapeutic compounds discussed below, which place them in a
separate category of estrogenic compounds distinguished from the
conventional physiological estrogens (e.g., estradiol and estrone).
These properties include low toxicity, long acting effect, and the
absence of any detectable carcinogenicity.
[0062] The estrogenic therapeutic compounds (particularly
estrogenic carboxylic acids) used in accordance with this invention
may also be a valuable research tool for testing the estrogen
receptors and/or pathways involved in mediating the actions
elicited by estrogenic and non-estrogenic compounds. As noted
earlier, there is an apparent activity/binding paradox suggesting
that the classic estrogen receptor, ER .alpha., may not be the
exclusive receptor or pathway mediating the actions of Z-BDDA
compounds, or possibly even those of estradiol. Initial studies
comparing the classical ER .alpha. and the novel estrogen receptor
ER .beta. show similar results. The binding affinity of (+)-Z-BDDA
is even lower than that of the (-)-enantiomer, and both enantiomers
have a much lower affinity for estrogen receptors than does
estradiol, where measured via direct receptor binding assays or by
generating does-response profiles using activation of
estrogen-responsive reportes genes in cell-culture systems. Indeed,
estradiol and other conventional estrogenic compounds appear to be
dependent on high affinity binding to ER .alpha. and/or .beta. for
eliciting the classical estrogen response. This effect does not
appear to be the case for the present estrogenic therapeutic
compounds (particularly the estrogenic carboxylic acids), which
suggests that these compounds are selective estrogen response
modulators (SERMs) that elicit their estrogenic actions by a novel
binding of the estrogen receptors, or by a mechanism that is
independent of the estrogen receptor and which occurs elsewhere in
the estrogen response pathway. Therefore, these estrogenic
therapeutic compounds (particularly estrogenic carboxylic acids)
have the potential for use as research tools in determining if a
classical or novel estrogenic action is dependent on high affinity
binding to ER .alpha. and/or .beta., or is elicited via low
affinity binding to the estrogen receptors. Additionally, the
present estrogenic compounds can be a valuable tool in elucidating
and characterizing the mechanisms involved in the classical and
novel estrogen signaling pathway, testing the estrogen receptors
and/or pathways involved in mediating the actions elicited by
estrogenic and non-estrogenic compounds. Sites of action of the
present estrogenic compounds can also be detected by
transcriptional initiation through a cotransfection assay.
[0063] A. Compounds
[0064] The estrogenic compounds useful in the methods of the
present invention include, for example, doisynolic acid compounds,
bisdehydrodoisynolic acid compounds, allenolic acid compounds,
phenyl- and hydroxyphenylcyclohexane compounds, phenyl- and
hydroxyphenyl-cyclohexene compounds, and
hydroxytetrahydroanthracene compounds. Especially preferred
compounds include, for example, doisynolic acid,
bisdehydrodoisynolic acid, allenolic acid, phenyl- and
hydroxyphenylcyclohexane carboxylic acids, phenyl- and
hydroxyphenyl-cyclohexene carboxylic acids, and
hydroxytetrahydroanthrace- ne carboxylic acids.
[0065] In addition to the foregoing specific compounds, a number of
derivatives thereof are also contemplated for use in the present
methods. These include hydroxynaphthyl alkylated alkanoic acids,
hydroxyphenyl alkylated cyclohexene- and cyclohexanecarboxylic
acids, and hydroxyalkylated tetrahydro-anthracenecarboxylic acids,
and the corresponding non-hydroxylated compounds which are
hydroxylated in vivo, and thereby likewise exhibit estrogenic
activity. Methods for preparing these derivatives involve
conventional synthetic organic chemical reactions within the
ordinary skill of the art.
[0066] In the various therapeutic methods disclosed herein, one can
use the estrogenically active compounds of the present invention in
their phenolic or non-phenolic forms, as free acids, or
corresponding pharmaceutically acceptable salts, ethers, esters, or
other easily hydrolyzable derivatives such as anhydrides, etc.
[0067] The preferred structures of the compounds for use in
accordance with this invention are shown below. It should be noted
that in some cases, only one enantiomeric structure is illustrated
for each of a particular compound. However, as these compounds
possess asymmetric carbon atoms, enantiomers and diastereomers
other than those shown may exhibit specific biological activity. As
it is known to those skilled in the art that the compounds of the
present invention having such asymmetric carbon atoms can exist in
enantiomeric, diastereomeric, and racemic forms, all these forms
are contemplated within the scope of the present invention. More
specifically, the present invention includes such enantiomers,
diastereomers, racemic mixtures, and other mixtures thereof.
[0068] In one of the more preferred embodiments of this invention,
the therapeutic estrogenic compound has the structure of formula
(I) or is a pharmaceutically acceptable salt thereof: 6
[0069] Here, X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6,
X.sup.7, X.sup.8, X.sup.9, and X.sup.10 preferably are carbon
atoms.
[0070] The dashed lines are optional .pi. bonds (i.e., a bond
represented by both a solid line and a dashed line may optionally
be either a single or a double bond).
[0071] R.sup.1.beta., R.sup.2.beta., R.sup.3.beta., R.sup.4.beta.,
R.sup.5, R.sup.6.beta., R.sup.7.beta., R.sup.8.beta.,
R.sup.9.beta., and/or R.sup.10 are present only when X.sup.1,
X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8,
X.sup.9, and/or X.sup.10, respectively, are saturated (i.e., are
bound to 4 atoms).
[0072] R.sup.1.alpha. and R.sup.2.alpha. preferably are
independently a moiety which (a) comprises from 1 to 20 carbon
atoms (more preferably from 1 to 6 carbon atoms), and is selected
from the group consisting of hydrocarbyl and substituted
hydrocarbyl (i.e., R.sup.1.alpha. and R.sup.2.alpha. are
independently a carbon-containing moiety which comprises no greater
than 20 carbon atoms, and more preferably no greater than 6 carbon
atoms); or (b) does not comprise a carbon atom and is selected from
the group consisting of amino, halogen, hydrogen, imino, nitro,
nitroso, oximido, oxo, oxy, phosphinidene, phosphino, phosphinyl,
phosphinylidene, phospho, phosphono, phosphoranyl,
phosphoranylidene, phosphoroso, siloxy, silyl, silylene, sulfeno,
sulfinyl, sulfino, sulfo, sulfonyl, thio, and thioxo. More
preferably, R.sup.1.alpha. and R.sup.2.alpha. are independently
selected from the group consisting of hydrogen; hydrocarbyl
comprising from 1 to 6 carbon atoms; and --OR.sup.100, wherein
R.sup.100 is hydrogen or hydrocarbyl containing from 1 to 6 carbon
atoms, and particularly wherein R.sup.100 is hydrogen or methyl.
Most preferably, R.sup.1.alpha. and R.sup.2.alpha. are
hydrogen.
[0073] R.sup.1.beta., R.sup.2.beta., R.sup.3.beta., R.sup.4.beta.,
R.sup.5, R.sup.6.beta., R.sup.8.beta., R.sup.9.beta., and R.sup.10
preferably are independently a moity which (a) comprises from 1 to
20 carbon atoms (more preferably from 1 to 6 carbon atoms), and is
selected from the group consisting of hydrocarbyl and substituted
hydrocarbyl; or (b) does not comprise a carbon atom and is selected
from the group consisting of amino, halogen, hydrogen, nitro,
nitroso, oxy, phosphino, phosphinyl, phospho, phosphono,
phosphoranyl, phosphoroso, siloxy, silyl, sulfeno, sulfino, sulfo,
sulfonyl, and thio. More preferably, R.sup.1.beta., R.sup.2.beta.,
R.sup.3.beta., R.sup.4.beta., R.sup.5, R.sup.6.beta.,
R.sup.8.beta., R.sup.9.beta., and R.sup.10 are independently
selected from the group consisting of hydrogen and hydrocarbyl
comprising from 1 to 6 carbon atoms. More preferably,
R.sup.1.beta., R.sup.2.beta., R.sup.3.beta., R.sup.4.beta.,
R.sup.5, R.sup.6.beta., R.sup.8.beta., R.sup.9.beta., and R.sup.10
are hydrogen.
[0074] R.sup.3.alpha. preferably (a) comprises from 1 to 20 carbon
atoms (more preferably from 1 to 6 carbon atoms), and is selected
from the group consisting of hydrocarbyl and substituted
hydrocarbyl; or (b) does not comprise a carbon atom and is selected
from the group consisting of amino, halogen, hydrogen, imino,
nitro, nitroso, oximido, oxo, oxy, phosphinidene, phosphino,
phosphinyl, phosphinylidene, phospho, phosphono, phosphoranyl,
phosphoranylidene, phosphoroso, siloxy, silyl, silylene, sulfeno,
sulfinyl, sulfino, sulfo, sulfonyl, thio, and thioxo. In a more
preferred embodiment, R.sup.3.alpha. is hydrogen. In another more
preferred embodiment, R.sup.3.alpha. is selected from the group
consisting of glycosidyl, acetylated glycosidyl, and malonylated
glycosidyl. In an additional more preferred embodiment,
R.sup.3.alpha. is --OC(O)(R.sup.101), wherein R.sup.101 is benzyl
or --N(CH.sub.2CH.sub.2Cl).sub.2. In yet another more preferred
embodiment, R.sup.3.alpha. comprises (a) no greater than 20 carbon
atoms (more preferably, no greater than 6 carbon atoms); and (b) a
moiety selected from the group consisting of amino, halogen,
hydrogen, imino, oximido, oxo, oxy, phosphinidene, phosphino,
phosphinyl, phosphinylidene, phosphono, phosphoranyl,
phosphoranylidene, siloxy, silyl, silylene, sulfeno, sulfino,
sulfo, and thio. In an even more preferred embodiment,
R.sup.3.alpha. comprises (a) no greater than 20 carbon atoms (more
preferably, no greater than 6 carbon atoms); and (b) a moiety
selected from the group consisting of amino, imino, oximido, oxy,
phosphinidene, phosphino, phosphinyl, phosphinylidene, phosphono,
phosphoranyl, phosphoranylidene, siloxy, silyl, silylene, sulfeno,
sulfino, sulfo, and thio. In a still even more preferred
embodiment, R.sup.3.alpha. comprises a polarizable hydrogen atom
(i.e., a hydrogen atom bound to an atom other than a carbon atom),
and is, for example, --C(O)(OH), --NH.sub.2, .dbd.NH, .dbd.N(OH),
--OH, .dbd.PH, --PH.sub.2, --P(O)(H)(H), .dbd.P(O)(H),
--P(O)(OH)(OH), --PH.sub.4, .dbd.PH.sub.3, .dbd.SiH.sub.2, --S(OH),
--S(O)(OH), S(O)(O)(OH), or --SH. In an alternative even more
preferred embodiment, R.sup.3.alpha. is --OR.sup.102 or
--OC(O)(R.sup.103), wherein R.sup.102 and R.sup.103 are hydrogen,
halogen, or hydrocarbyl comprising from 1 to 19 carbon atoms
(particularly 1 to 5 carbon atoms, and more particularly from 1 to
2 carbon atoms). Most preferably, R.sup.3.alpha. is --OH.
[0075] R.sup.4.alpha., R.sup.6.alpha., and R.sup.9.alpha.
preferably are independently a moiety which (a) comprises from 1 to
20 carbon atoms (more preferably from 1 to 6 carbon atoms), and is
selected from the group consisting of hydrocarbyl and substituted
hydrocarbyl; or (b) does not comprise a carbon atom and is selected
from the group consisting of amino, halogen, hydrogen, imino,
nitro, nitroso, oximido, oxo, oxy, phosphinidene, phosphino,
phosphinyl, phosphinylidene, phospho, phosphono, phosphoranyl,
phosphoranylidene, phosphoroso, siloxy, silyl, silylene, sulfeno,
sulfinyl, sulfino, sulfo, sulfonyl, thio, and thioxo. More
preferably, R.sup.4.alpha., R.sup.6.alpha., and R.sup.9.alpha. are
independently selected from the group consisting of hydrogen and
hydrocarbyl comprising from 1 to 6 carbon atoms. Most preferably,
R.sup.4.alpha., R.sup.6.alpha., and R.sup.9.alpha. are
hydrogen.
[0076] R.sup.7.alpha. preferably (a) comprises from 1 to 20 carbon
atoms (more preferably from 1 to 6 carbon atoms), and is selected
from the group consisting of hydrocarbyl and substituted
hydrocarbyl; or (b) does not comprise a carbon atom and is selected
from the group consisting of amino, halogen, hydrogen, imino,
nitro, nitroso, oximido, oxo, oxy, phosphinidene, phosphino,
phosphinyl, phosphinylidene, phospho, phosphono, phosphoranyl,
phosphoranylidene, phosphoroso, siloxy, silyl, silylene, sulfeno,
sulfinyl, sulfino, sulfo, sulfonyl, thio, and thioxo. More
preferably, R.sup.7.alpha. is selected from the group consisting of
hydrogen, hydrocarbyl comprising from 1 to 20 carbon atoms, and
oxo. Most preferably, R.sup.7.alpha. is hydrogen.
[0077] R.sup.7.beta. preferably (a) comprises from 1 to 20 carbon
atoms (more preferably from 1 to 6 carbon atoms), and is selected
from the group consisting of hydrocarbyl and substituted
hydrocarbyl; or (b) does not comprise a carbon atom and is selected
from the group consisting of amino, halogen, hydrogen, nitro,
nitroso, oxy, phosphino, phosphinyl, phospho, phosphono,
phosphoranyl, phosphoroso, siloxy, silyl, sulfeno, sulfino, sulfo,
sulfonyl, and thio. More preferably, R.sup.7.beta. is selected from
the group consisting of hydrogen and hydrocarbyl comprising from 1
to 6 carbon atoms. Most preferably, R.sup.7.beta. is hydrogen.
[0078] R.sup.8.alpha. preferably is a substituted hydrocarbyl
comprising a moiety selected from the group consisting of amino,
imino, oximido, oxy, phosphinidene, phosphino, phosphinyl,
phosphinylidene, phosphono, phosphoranyl, phosphoranylidene,
siloxy, silyl, silylene, sulfeno, sulfino, sulfo, and thio. More
preferably, R.sup.8.alpha. comprises a substituted hydrocarbyl
containing a polarizable hydrogen atom (R.sup.8.alpha. is, for
example, --C(O)(OH), --NH.sub.2, .dbd.NH, .dbd.N(OH), --OH,
.dbd.PH, --PH.sub.2, --P(O)(H)(H), .dbd.P(O)(H), --P(O)(OH)(OH),
--PH.sub.4, .dbd.PH.sub.3, .dbd.SiH.sub.2, --S(OH), --S(O)(OH),
S(O)(O)(OH), or --SH). In an alternative more preferred embodiment,
R.sup.8.alpha. is a substituted hydrocarbyl comprising
--C(O)(OR.sup.104), wherein R.sup.104 is hydrogen, halogen, or
hydrocarbyl comprising from 1 to 5 carbon atoms (particularly from
1 to 2 carbon atoms). Most preferably, R.sup.8.alpha. is a
substituted hydrocarbyl comprising --C(O)(OH), making the compound
an estrogenic carboxylic acid. In a particularly preferred
embodiment, R.sup.8.alpha. comprises no greater than 20 carbon
atoms.
[0079] In some instances, it is preferred that the therapeutic
compound of formula (I) have the structure of formula (II) or be a
pharmaceutically acceptable salt thereof: 7
[0080] Here, X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6,
X.sup.7, X.sup.8, X.sup.9, X.sup.10, R.sup.1.alpha., R.sup.1.beta.,
R.sup.2.alpha., R.sup.2.beta., R.sup.3.alpha., R.sup.3.beta.,
R.sup.4.alpha., R.sup.4.beta., R.sup.5, R.sup.6.alpha.,
R.sup.6.beta., R.sup.7.alpha., R.sup.7.beta., R.sup.8.beta.,
R.sup.9.alpha., R.sup.9.beta., and R.sup.10 are preferably as
defined above for formula (I).
[0081] X.sup.13 and X.sup.14 preferably are carbon atoms.
[0082] The dashed lines are optional .pi. bonds.
[0083] R.sup.1.beta., R.sup.2.beta., R.sup.3.beta., R.sup.4.beta.,
R.sup.5, R.sup.6.beta., R.sup.7.beta., R.sup.8.beta.,
R.sup.9.beta., R.sup.10, R.sup.13.gamma., and/or R.sup.14.beta. are
present only when X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5,
X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.13, and/or
X.sup.14, respectively, are saturated.
[0084] R.sup.13.alpha. preferably comprises (a) no greater than 20
carbon atoms (more preferably no greater than 6 carbon atoms); and
(b) a moiety selected from the group consisting of amino, imino,
oximido, oxy, phosphinidene, phosphino, phosphinyl,
phosphinylidene, phosphono, phosphoranyl, phosphoranylidene,
siloxy, silyl, silylene, sulfeno, sulfino, sulfo, and thio. More
preferably, R.sup.13.alpha. comprises a polarizable hydrogen atom
and is, for example, --C(O)(OH), --NH.sub.2, .dbd.NH, .dbd.N(OH),
--OH, .dbd.PH, --PH.sub.2, --P(O)(H)(H), .dbd.P(O)(H),
--P(O)(OH)(OH), --PH.sub.4, .dbd.PH.sub.3, .dbd.SiH.sub.2, --S(OH),
--S(O)(OH), S(O)(O)(OH), or --SH. In an alternative more preferred
embodiment, R.sup.13.alpha. is --C(O)(OR.sup.105), wherein
R.sup.105 is hydrogen, halogen, or hydrocarbyl comprising from 1 to
5 carbon atoms. Most preferably, R.sup.13.alpha. is --C(O)(OH)
(i.e., the compound is an estrogenic carboxylic acid).
[0085] R.sup.13.beta. preferably (a) comprises from 1 to 20 carbon
atoms (more preferably no greater than 6 carbon atoms), and is
selected from the group consisting of hydrocarbyl and substituted
hydrocarbyl; or (b) does not comprise a carbon atom and is selected
from the group consisting of amino, halogen, hydrogen, imino,
nitro, nitroso, oximido, oxo, oxy, phosphinidene, phosphino,
phosphinyl, phosphinylidene, phospho, phosphono, phosphoranyl,
phosphoranylidene, phosphoroso, siloxy, silyl, silylene, sulfeno,
sulfinyl, sulfino, sulfo, sulfonyl, thio, and thioxo. More
preferably, R.sup.13.beta. is selected from the group consisting of
hydrogen and hydrocarbyl comprising from 1 to 6 carbon atoms. Even
more preferably, R.sup.13.beta. is are independently hydrocarbyl
comprising from 1 to 6 carbon atoms. Most preferably,
R.sup.13.beta. is methyl.
[0086] R.sup.13.gamma. preferably (a) comprises from 1 to 20 carbon
atoms (more preferably no greater than 6 carbon atoms), and is
selected from the group consisting of hydrocarbyl and substituted
hydrocarbyl; or (b) does not comprise a carbon atom and is selected
from the group consisting of amino, halogen, hydrogen, nitro,
nitroso, oxy, phosphino, phosphinyl, phospho, phosphono,
phosphoranyl, phosphoroso, siloxy, silyl, sulfeno, sulfino, sulfo,
sulfonyl, and thio. More preferably, R.sup.13.gamma. is selected
from the group consisting of hydrogen and hydrocarbyl comprising
from 1 to 6 carbon atoms. Even more preferably, R.sup.13.gamma. is
hydrocarbyl comprising from 1 to 6 carbon atoms. Most preferably,
R.sup.13.gamma. is methyl.
[0087] R.sup.14.alpha. preferably (a) comprises from 1 to 20 carbon
atoms (more preferably no greater than 6 carbon atoms), and is
selected from the group consisting of hydrocarbyl and substituted
hydrocarbyl; or (b) does not comprise a carbon atom and is selected
from the group consisting of amino, halogen, hydrogen, imino,
nitro, nitroso, oximido, oxo, oxy, phosphinidene, phosphino,
phosphinyl, phosphinylidene, phospho, phosphono, phosphoranyl,
phosphoranylidene, phosphoroso, siloxy, silyl, silylene, sulfeno,
sulfinyl, sulfino, sulfo, sulfonyl, thio, and thioxo. More
preferably, R.sup.14.alpha. is selected from the group consisting
of hydrogen and hydrocarbyl comprising from 1 to 6 carbon atoms.
Even more preferably, R.sup.14.alpha. is hydrocarbyl comprising
from 1 to 6 carbon atoms. Most preferably, R.sup.14.alpha. is
ethyl.
[0088] R.sup.14.beta. preferably (a) comprises from 1 to 20 (more
preferably no greater than 6 carbon atoms), carbon atoms and is
selected from the group consisting of hydrocarbyl and substituted
hydrocarbyl; or (b) does not comprise a carbon atom and is selected
from the group consisting of amino, halogen, hydrogen, nitro,
nitroso, oxy, phosphino, phosphinyl, phospho, phosphono,
phosphoranyl, phosphoroso, siloxy, silyl, sulfeno, sulfino, sulfo,
sulfonyl, and thio. More preferably, R.sup.14.beta. is selected
from the group consisting of hydrogen and hydrocarbyl comprising
from 1 to 6 carbon atoms. Most preferably, R.sup.14.beta. is
hydrogen.
[0089] One particularly preferred structure of formula (II) is
"allenolic acid" (or "AA"), which has the following formula (M):
8
[0090] In other instances, it is particularly preferred for
R.sup.8.alpha. to form a carbocyclic ring with R.sup.9.alpha. in
formula (I) to create, for example, a doisynolic acid derivative
having the formula (IV) or a pharmaceutically acceptable salt
thereof: 9
[0091] Here, X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6,
X.sup.7, X.sup.8, X.sup.9, X.sup.10, R.sup.1.alpha., R.sup.1.beta.,
R.sup.2.alpha., R.sup.2.beta., R.sup.3.alpha., R.sup.3.beta.,
R.sup.4.alpha., R.sup.4.beta., R.sup.5, R.sup.6.alpha.,
R.sup.6.beta., R.sup.7.alpha., R.sup.7.beta., R.sup.8.beta.,
R.sup.9.beta., and R.sup.10 are preferably as defined above for
formula (I).
[0092] X.sup.11, X.sup.12, X.sup.13 and X.sup.14 preferably are
carbon atoms.
[0093] The dashed lines are optional .pi. bonds.
[0094] R.sup.1.beta., R.sup.2.beta., R.sup.3.beta., R.sup.4.beta.,
R.sup.5, R.sup.6.beta., R.sup.7.beta., R.sup.8.beta.,
R.sup.9.beta., R.sup.10, R.sup.11.beta., R.sup.12.beta.,
R.sup.13.beta., and/or R.sup.14.beta. are present only when
X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7,
X.sup.8, X.sup.9, X.sup.10, X.sup.13, and/or X.sup.14,
respectively, are saturated.
[0095] R.sup.11.alpha. and R.sup.12.alpha. preferably are
independently a moiety which (a) comprises from 1 to 20 carbon
atoms (more preferably, from 1 to 6 carbon atoms), and is selected
from the group consisting of hydrocarbyl and substituted
hydrocarbyl; or (b) does not comprise a carbon atom and is selected
from the group consisting of amino, halogen, hydrogen, imino,
nitro, nitroso, oximido, oxo, oxy, phosphinidene, phosphino,
phosphinyl, phosphinylidene, phospho, phosphono, phosphoranyl,
phosphoranylidene, phosphoroso, siloxy, silyl, silylene, sulfeno,
sulfinyl, sulfino, sulfo, sulfonyl, thio, and thioxo. More
preferably, R.sup.11.alpha. and R.sup.12.alpha. are independently
selected from the group consisting of hydrogen and hydrocarbyl
comprising from 1 to 6 carbon atoms. Most preferably,
R.sup.11.alpha. and R.sup.12.alpha. are hydrogen.
[0096] R.sup.11.beta., R.sup.12.beta., and R.sup.14.beta.
preferably are independently a moiety which (a) comprises from 1 to
20 carbon atoms (more preferably, from 1 to 6 carbon atoms), and is
selected from the group consisting of hydrocarbyl and substituted
hydrocarbyl; or (b) does not comprise a carbon atom and is selected
from the group consisting of amino, halogen, hydrogen, nitro,
nitroso, oxy, phosphino, phosphinyl, phospho, phosphono,
phosphoranyl, phosphoroso, siloxy, silyl, sulfeno, sulfino, sulfo,
sulfonyl, and thio. More preferably, R.sup.11.beta.,
R.sup.12.beta., and R.sup.14.beta. are independently selected from
the group consisting of hydrogen and hydrocarbyl comprising from 1
to 6 carbon atoms. Most preferably, R.sup.11.beta., R.sup.12.beta.,
and R.sup.14.beta. are hydrogen.
[0097] R.sup.13.alpha. preferably comprises (a) no greater than 20
carbon atoms (more preferably no greater than 6 carbon atoms); and
(b) a moiety selected from the group consisting of amino, imino,
oximido, oxy, phosphinidene, phosphino, phosphinyl,
phosphinylidene, phosphono, phosphoranyl, phosphoranylidene,
siloxy, silyl, silylene, sulfeno, sulfino, sulfo, and thio. More
preferably, R.sup.13.alpha. comprises a polarizable hydrogen atom
and is, for example, --C(O)(OH), --NH.sub.2, NH, .dbd.N(OH), --OH,
.dbd.PH, --PH.sub.2, --P(O)(H)(H), .dbd.P(O)(H), --P(O)(OH)(OH),
--PH.sub.4, .dbd.PH.sub.3, .dbd.SiH.sub.2, --S(OH), --S(O)(OH),
S(O)(O)(OH), or --SH. In an alternative more preferred embodiment,
R.sup.13.alpha. is --C(O)(OR.sup.106), wherein R.sup.106 is
hydrogen, halogen, or hydrocarbyl comprising from 1 to 5 carbon
atoms. Most preferably, R.sup.13.alpha. is --C(O)(OH) (i.e., the
compound is an estrogenic carboxylic acid).
[0098] R.sup.13.beta. preferably (a) comprises from 1 to 20 carbon
atoms and is selected from the group consisting of hydrocarbyl and
substituted hydrocarbyl; or (b) does not comprise a carbon atom and
is selected from the group consisting of amino, halogen, hydrogen,
nitro, nitroso, oxy, phosphino, phosphinyl, phospho, phosphono,
phosphoranyl, phosphoroso, siloxy, silyl, sulfeno, sulfino, sulfo,
sulfonyl, and thio. More preferably, R.sup.13.beta. is selected
from the group consisting of hydrogen and hydrocarbyl comprising
from 1 to 6 carbon atoms. Even more preferably, R.sup.13.beta. is
hydrocarbyl comprising from 1 to 6 carbon atoms. Most preferably,
R.sup.13.beta. is methyl.
[0099] R.sup.14.alpha. preferably (a) comprises from 1 to 20 carbon
atoms and is selected from the group consisting of hydrocarbyl and
substituted hydrocarbyl; or (b) does not comprise a carbon atom and
is selected from the group consisting of amino, halogen, hydrogen,
imino, nitro, nitroso, oximido, oxo, oxy, phosphinidene, phosphino,
phosphinyl, phosphinylidene, phospho, phosphono, phosphoranyl,
phosphoranylidene, phosphoroso, siloxy, silyl, silylene, sulfeno,
sulfinyl, sulfino, sulfo, sulfonyl, thio, and thioxo. More
preferably, R.sup.14.alpha. is selected from the group consisting
of hydrogen and hydrocarbyl comprising from 1 to 6 carbon atoms.
Even more preferably, R.sup.14.alpha. is hydrocarbyl comprising
from 1 to 6 carbon atoms. Most preferably, R.sup.14.alpha. is
ethyl.
[0100] It is especially preferred for the A ring of formula (IV) to
be aromatic, as shown in formula (V): 10
[0101] Here, X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6,
X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13,
X.sup.14, R.sup.6.alpha., R.sup.6.beta., R.sup.7.alpha.,
R.sup.7.beta., R.sup.8.beta., R.sup.9.beta., R.sup.11.alpha.,
R.sup.11.beta., R.sup.12.alpha., R.sup.12.beta., R.sup.13.alpha.,
R.sup.13.beta., R.sup.14.alpha., and R.sup.14.beta. preferably are
as defined above for formula (IV).
[0102] The dashed lines are optional .pi. bonds.
[0103] R.sup.6.beta., R.sup.7.beta., R.sup.8.beta., R.sup.9.beta.,
R.sup.11.beta., R.sup.12.beta., R.sup.13.beta., and/or
R.sup.14.beta. are present only when X.sup.6, X.sup.7, X.sup.8,
X.sup.9, X.sup.11, X.sup.12, X.sup.13, and/or X.sup.14,
respectively, are saturated.
[0104] R.sup.1.alpha. and R.sup.2.alpha. preferably are
independently a moiety which (a) comprises from 1 to 20 carbon
atoms (more preferably from 1 to 6 carbon atoms), and is selected
from the group consisting of hydrocarbyl and substituted
hydrocarbyl; or (b) does not comprise a carbon atom and is selected
from the group consisting of amino, halogen, hydrogen, nitro,
nitroso, oxy, phosphino, phosphinyl, phospho, phosphono,
phosphoranyl, phosphoroso, siloxy, silyl, sulfeno, sulfino, sulfo,
sulfonyl, and thio. More preferably, R.sup.1.alpha. and
R.sup.2.alpha. are independently selected from the group consisting
of hydrogen; hydrocarbyl comprising from 1 to 6 carbon atoms; and
--OR.sup.107, wherein R.sup.107 is hydrogen or hydrocarbyl
containing from 1 to 6 carbon atoms, and particularly wherein
R.sup.107 is hydrogen or methyl. Most preferably, R.sup.1.alpha.
and R.sup.2.alpha. are hydrogen.
[0105] R.sup.3.alpha. preferably (a) comprises from 1 to 20 carbon
atoms (more preferably from 1 to 6 carbon atoms), and is selected
from the group consisting of hydrocarbyl and substituted
hydrocarbyl; or (b) does not comprise a carbon atom and is selected
from the group consisting of amino, halogen, hydrogen, nitro,
nitroso, oxy, phosphino, phosphinyl, phospho, phosphono,
phosphoranyl, phosphoroso, siloxy, silyl, sulfeno, sulfino, sulfo,
sulfonyl, and thio. In a more preferred embodiment, R.sup.3.alpha.
is hydrogen. In another more preferred embodiment, R.sup.3.alpha.
is selected from the group consisting of glycosidyl, acetylated
glycosidyl, and malonylated glycosidyl. In an additional more
preferred embodiment, R.sup.3.alpha. is --OC(O)(R.sup.108), wherein
R.sup.108 is benzyl or --N(CH.sub.2CH.sub.2Cl).sub.2. In yet
another more preferred embodiment, R.sup.3.alpha. comprises (a) no
greater than 20 carbon atoms (more preferably, no greater than 6
carbon atoms); and (b) a moiety selected from the group consisting
of amino, imino, oximido, oxy, phosphinidene, phosphino,
phosphinyl, phosphinylidene, phosphono, phosphoranyl,
phosphoranylidene, siloxy, silyl, silylene, sulfeno, sulfino,
sulfo, and thio. In an even more preferred embodiment,
R.sup.3.alpha. comprises a polarizable hydrogen atom and is, for
example, --C(O)(OH), --NH.sub.2, .dbd.NH, .dbd.N(OH), --OH,
.dbd.PH, --PH.sub.2, --P(O)(H)(H), .dbd.P(O)(H), --P(O)(OH)(OH),
--PH.sub.4, .dbd.PH.sub.3, .dbd.SiH.sub.2, --S(OH), --S(O)(OH),
S(O)(O)(OH), or --SH. In an alternative even more preferred
embodiment, R.sup.3.alpha. is --OR.sup.109 or --OC(O)R.sup.110,
wherein R.sup.109 and R.sup.110 are hydrogen, halogen, or
hydrocarbyl comprising from 1 to 19 carbon atoms (particularly 1 to
5 carbon atoms, and more particularly 1 to 2 carbon atoms). Most
preferably, R.sup.3.alpha. is --OH.
[0106] R.sup.4.alpha. preferably (a) comprises from 1 to 20 carbon
atoms (more preferably from 1 to 6 atoms), and is selected from the
group consisting of hydrocarbyl and substituted hydrocarbyl; or (b)
does not comprise a carbon atom and is selected from the group
consisting of amino, halogen, hydrogen, nitro, nitroso, oxy,
phosphino, phosphinyl, phospho, phosphono, phosphoranyl,
phosphoroso, siloxy, silyl, sulfeno, sulfino, sulfo, sulfonyl, and
thio. More preferably, R.sup.4.alpha. is selected from the group
consisting of hydrogen and hydrocarbyl comprising from 1 to 6
carbon atoms. Most preferably, R.sup.4.alpha. is hydrogen.
[0107] In another especially preferred embodiment using the
compound of formula (V) or a pharmaceutically acceptable salt
thereof, no .pi. bonds exist in the bond positions represented by
both a solid line and a dashed line (i.e., all the bonds in those
positions are single bonds) in formula (V), and the compound
consequently has formula (VI) or is a pharmaceutically acceptable
salt thereof: 11
[0108] Here, X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6,
X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13,
X.sup.14, R.sup.1.alpha., R.sup.2.alpha., R.sup.3.alpha.,
R.sup.4.alpha., R.sup.6.alpha., R.sup.6.beta., R.sup.7.alpha.,
R.sup.7.beta., R.sup.8.beta., R.sup.9.beta., R.sup.11.alpha.,
R.sup.11.beta., R.sup.12.alpha., R.sup.12.beta., R.sup.13.alpha.,
R.sup.13.beta., R.sup.14.alpha., and R.sup.14.beta. preferably are
as defined for formula (V).
[0109] R.sup.6.beta., R.sup.7.beta., R.sup.11.beta.,
R.sup.12.beta., R.sup.13.beta., and R.sup.14.beta. are present only
when X.sup.6, X.sup.7, X.sup.11, X.sup.12, X.sup.13, and/or
X.sup.14, respectively, are saturated.
[0110] When using the compound of formula (VI) or a
pharmaceutically acceptable salt thereof, it is especially
preferred for R.sup.1.alpha., R.sup.2.alpha., R.sup.4.alpha.,
R.sup.6.alpha., R.sup.6.beta., R.sup.7.alpha., R.sup.7.beta.,
R.sup.8.beta., R.sup.9.beta., R.sup.11.alpha., R.sup.11.beta.,
R.sup.12.alpha., R.sup.12.beta., and R.sup.14.beta. to be hydrogen;
R.sup.3.alpha. to be --OH; R.sup.13.alpha. to be --C(O)(OH)(i.e.,
the compound is an estrogenic carboxylic acid); R.sup.13.beta. to
be methyl; and R.sup.14.alpha. to be ethyl. Such a compound most
preferably has the formula (VII) or is a pharmaceutically
acceptable salt thereof: 12
[0111] In another particularly preferred embodiment, the compound
of formula (V) or the pharmaceutically acceptable salt thereof has
the formula (VIII) or is a pharmaceutically acceptable salt
thereof: 13
[0112] Here, X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6,
X.sup.7, X.sup.8, X.sup.9, X.sup.10, X.sup.11, X.sup.9, X.sup.10,
X.sup.11, X.sup.12, X.sup.13, X.sup.14, R.sup.1.alpha.,
R.sup.2.alpha., R.sup.3.alpha., R.sup.4.alpha., R.sup.11.alpha.,
R.sup.11.beta., R.sup.12.alpha., R.sup.12.beta., R.sup.13.alpha.,
R.sup.13.beta., R.sup.14.alpha., and R.sup.4.beta. preferably are
as defined for formula (VI.
[0113] R.sup.11.beta., R.sup.12.beta., R.sup.13.beta., and/or
R.sup.14.beta. are present only when X.sup.11, X.sup.12, X.sup.13,
and/or X.sup.14, respectively, are saturated.
[0114] R.sup.6.alpha. preferably (a) comprises from 1 to 20 carbon
atoms (more preferably from 1 to 6 carbon atoms), and is selected
from the group consisting of hydrocarbyl and substituted
hydrocarbyl; or (b) does not comprise a carbon atom and is selected
from the group consisting of amino, halogen, hydrogen, nitro,
nitroso, oxy, phosphino, phosphinyl, phospho, phosphono,
phosphoranyl, phosphoroso, siloxy, silyl, sulfeno, sulfino, sulfo,
sulfonyl, and thio. More preferably, R.sup.6.alpha. is selected
from the group consisting of hydrogen and hydrocarbyl comprising
from 1 to 6 carbon atoms. Most preferably, R.sup.6.alpha. is
hydrogen.
[0115] R.sup.7.alpha. preferably (a) comprises from 1 to 20 carbon
atoms (more preferably from 1 to 6 carbon atoms), and is selected
from the group consisting of hydrocarbyl and substituted
hydrocarbyl; or (b) does not comprise a carbon atom and is selected
from the group consisting of amino, halogen, hydrogen, nitro,
nitroso, oxy, phosphino, phosphinyl, phospho, phosphono,
phosphoranyl, phosphoroso, siloxy, silyl, sulfeno, sulfino, sulfo,
sulfonyl, and thio. More preferably, R.sup.7.alpha. is selected
from the group consisting of hydrogen and hydrocarbyl comprising
from 1 to 20 carbon atoms. Most preferably, R.sup.7.alpha. is
hydrogen.
[0116] When the compound has formula (VIII), it is especially
preferred for R.sup.1.alpha., R.sup.2.alpha., R.sup.4.alpha.,
R.sup.6.alpha., R.sup.7.alpha., R.sup.11.alpha., R.sup.11.beta.,
R.sup.12.alpha., R.sup.12.beta., and R.sup.14.beta. to be hydrogen;
R.sup.3.alpha. to be --OH; R.sup.13.alpha. to be --C(O)(OH) (i.e.,
the compound is an estrogenic carboxylic acid); R.sup.13 to be
methyl; and R.sup.14.alpha. to be ethyl. Such a compound, for
example, may have the formula (IX): 14
[0117] This compound is sometimes described herein as
"(-)-Z-bisdehydrodoisynolic acid" (or "(-)-Z-BDDA"). Its enantiomer
has the formula (X): 15
[0118] This compound is sometimes described herein as
"(+)-Z-bisdehydrodoisynolic acid" (or "(+)-Z-BDDA"). Depending on
the therapeutic application, either formula (IX) or formula (X) is
most preferred.
[0119] In some instances, it is particularly preferred for
R.sup.8.alpha. in Formula (I) to form a carbocyclic ring with
R.sup.7.alpha. to form a compound having the formula (XI) or a
pharmaceutically acceptable salt thereof: 16
[0120] Here, X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6,
X.sup.7, X.sup.8, X.sup.9, X.sup.10, R.sup.1.alpha., R.sup.1.beta.,
R.sup.2.alpha., R.sup.2.beta., R.sup.3.alpha., R.sup.3.beta.,
R.sup.4.alpha., R.sup.4.beta., R.sup.5, R.sup.6.alpha.,
R.sup.6.beta., R.sup.7.beta., R.sup.8.beta., R.sup.9.alpha.,
R.sup.9.beta., and R.sup.10 preferably are as defined above for
formula (I).
[0121] R.sup.1.beta., R.sup.2.beta., R.sup.3.beta., R.sup.4.beta.,
R.sup.5, R.sup.6.beta., R.sup.7.beta., R.sup.8.beta.,
R.sup.9.beta., R.sup.10, R.sup.14.beta., R.sup.20.beta.,
R.sup.21.beta., and/or R.sup.22.beta., respectively, are
saturated.
[0122] R.sup.21.alpha. and R.sup.22.alpha. preferably are
independently a moiety which (a) comprises from 1 to 20 carbon
atoms (more preferably, from 1 to 6 carbon atoms), and is selected
from the group consisting of hydrocarbyl and substituted
hydrocarbyl; or (b) does not comprise a carbon atom and is selected
from the group consisting of amino, halogen, hydrogen, imino,
nitro, nitroso, oximido, oxo, oxy, phosphinidene, phosphino,
phosphinyl, phosphinylidene, phospho, phosphono, phosphoranyl,
phosphoranylidene, phosphoroso, siloxy, silyl, silylene, sulfeno,
sulfinyl, sulfino, sulfo, sulfonyl, thio, and thioxo. More
preferably, R.sup.21.alpha. and R.sup.22.alpha. are independently
selected from the group consisting of hydrogen and hydrocarbyl
comprising from 1 to 6 carbon atoms. Most preferably,
R.sup.21.alpha. and R.sup.22.alpha. are hydrogen.
[0123] R.sup.21.beta., R.sup.22.beta., and R.sup.14.beta.
preferably are independently a moiety which (a) comprises from 1 to
20 carbon atoms (more preferably, from 1 to 6 carbon atoms), and is
selected from the group consisting of hydrocarbyl and substituted
hydrocarbyl; or (b) does not comprise a carbon atom and is selected
from the group consisting of amino, halogen, hydrogen, nitro,
nitroso, oxy, phosphino, phosphinyl, phospho, phosphono,
phosphoranyl, phosphoroso, siloxy, silyl, sulfeno, sulfino, sulfo,
sulfonyl, and thio. More preferably, R.sup.21.beta.,
R.sup.22.beta., and R.sup.14.beta. are independently selected from
the group consisting of hydrogen and hydrocarbyl comprising from 1
to 6 carbon atoms. Most preferably, R.sup.21.beta., R.sup.22.beta.,
and R.sup.14.beta. are hydrogen.
[0124] R.sup.20.alpha. preferably comprises (a) no greater than 20
carbon atoms (more preferably no greater than 6 carbon atoms); and
(b) a moiety selected from the group consisting of amino, imino,
oximido, oxy, phosphinidene, phosphino, phosphinyl,
phosphinylidene, phosphono, phosphoranyl, phosphoranylidene,
siloxy, silyl, silylene, sulfeno, sulfino, sulfo, and thio. More
preferably, R.sup.20.alpha. comprises a polarizable hydrogen atom
and is, for example, --C(O)(OH), --NH.sub.2, .dbd.NH, .dbd.N(OH),
--OH, .dbd.PH, --PH.sub.2, --P(O)(H)(H), .dbd.P(O)(H),
--P(O)(OH)(OH), --PH.sub.4, .dbd.PH.sub.3, .dbd.SiH.sub.2, --S(OH),
--S(O)(OH), S(O)(O)(OH), or --SH. In an alternative more preferred
embodiment, R.sup.20.alpha. is --C(O)(OR.sup.111), wherein
R.sup.111 is hydrogen, halogen, or hydrocarbyl comprising from 1 to
5 carbon atoms. Most preferably, R.sup.20.alpha. is --C(O)(OH)
(i.e., the compound is an estrogenic carboxylic acid).
[0125] R.sup.20.beta. preferably (a) comprises from 1 to 20 carbon
atoms and is selected from the group consisting of hydrocarbyl and
substituted hydrocarbyl; or (b) does not comprise a carbon atom and
is selected from the group consisting of amino, halogen, hydrogen,
nitro, nitroso, oxy, phosphino, phosphinyl, phospho, phosphono,
phosphoranyl, phosphoroso, siloxy, silyl, sulfeno, sulfino, sulfo,
sulfonyl, and thio. More preferably, R.sup.20.beta. is selected
from the group consisting of hydrogen and hydrocarbyl comprising
from 1 to 6 carbon atoms. Even more preferably, R.sup.20.beta. is
hydrocarbyl comprising from 1 to 6 carbon atoms. Most preferably,
R.sup.20.beta. is methyl.
[0126] R.sup.14.alpha. preferably (a) comprises from 1 to 20 carbon
atoms and is selected from the group consisting of hydrocarbyl and
substituted hydrocarbyl; or (b) does not comprise a carbon atom and
is selected from the group consisting of amino, halogen, hydrogen,
imino, nitro, nitroso, oximido, oxo, oxy, phosphinidene, phosphino,
phosphinyl, phosphinylidene, phospho, phosphono, phosphoranyl,
phosphoranylidene, phosphoroso, siloxy, silyl, silylene, sulfeno,
sulfinyl, sulfino, sulfo, sulfonyl, thio, and thioxo. More
preferably, R.sup.14.alpha. is selected from the group consisting
of hydrogen and hydrocarbyl comprising from 1 to 6 carbon atoms.
Even more preferably, R.sup.14.alpha. is hydrocarbyl comprising
from 1 to 6 carbon atoms. Most preferably, R.sup.14.alpha. is
ethyl.
[0127] When the compound has formula (XI), it is particularly
preferred for the A and B rings to be aromatic; R.sup.1.alpha.,
R.sup.2.alpha., R.sup.4.alpha., R.sup.6.alpha., R.sup.9.alpha.,
R.sup.14.beta., R.sup.21.alpha., R.sup.21.beta., R.sup.22.alpha.,
and R.sup.2.beta. to be hydrogen; R.sup.3.alpha. to be --OH;
R.sup.14.alpha., to be ethyl; R.sup.20.alpha. to be --C(O)(OH)
(i.e., the compound is an estrogenic carboxylic acid);
R.sup.20.beta. to be methyl. Most preferably, such a compound is
formula (XII) or a pharmaceutically acceptable salt thereof: 17
[0128] This compound is sometimes referred to herein as
1-ethyl-6-hydroxy-2-methyl-1,2,3,4-tetrahydroanthracene-2-carboxylic
acid.
[0129] In another of the more preferred embodiments of this
invention, the compound has the structure of formula (XIII) or is a
pharmaceutically acceptable salt thereof: 18
[0130] Here, X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.8,
X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and X.sup.14
preferably are carbon atoms.
[0131] The dashed lines are optional .pi. bonds.
[0132] R.sup.1.beta., R.sup.2.beta., R.sup.3.beta., R.sup.4.beta.,
R.sup.5.beta., R.sup.8.beta., R.sup.9, R.sup.10, R.sup.11.beta.,
R.sup.12.beta., R.sup.13.beta., and/or R.sup.14.beta. are present
only when X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.8,
X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, and/or X.sup.14,
respectively, are saturated.
[0133] R.sup.1.alpha. and R.sup.2.alpha. preferably are
independently a moiety which (a) comprises from 1 to 20 carbon
atoms (more preferably from 1 to 6 carbon atoms), and is selected
from the group consisting of hydrocarbyl and substituted
hydrocarbyl; or (b) does not comprise a 10 carbon atom and is
selected from the group consisting of amino, halogen, hydrogen,
imino, nitro, nitroso, oximido, oxo, oxy, phosphinidene, phosphino,
phosphinyl, phosphinylidene, phospho, phosphono, phosphoranyl,
phosphoranylidene, phosphoroso, siloxy, silyl, silylene, sulfeno,
sulfinyl, sulfino, sulfo, sulfonyl, thio, and thioxo. More
preferably, R.sup.1.alpha. and R.sup.2.alpha. are independently
selected from the group consisting of hydrogen; hydrocarbyl 15
comprising from 1 to 6 carbon atoms; and --OR.sup.112, wherein
R.sup.112 is hydrogen or hydrocarbyl containing from 1 to 6 carbon
atoms, and particularly wherein R.sup.112 is hydrogen or methyl.
Most preferably, R.sup.1.alpha. and R.sup.2.alpha. are
hydrogen.
[0134] R.sup.1.beta., R.sup.2.beta., R.sup.3.beta., R.sup.4.beta.,
R.sup.5.beta., R.sup.8.beta., R.sup.9, R.sup.10, R.sup.11.beta.,
and R.sup.13.beta. preferably are independently a moiety which (a)
comprises from 1 to 20 carbon atoms (more preferably from 1 to 6
carbon atoms), and is selected from the group consisting of
hydrocarbyl and substituted hydrocarbyl; or (b) does not comprise a
carbon atom and is selected from the group consisting of amino,
halogen, hydrogen, nitro, nitroso, oxy, phosphino, phosphinyl,
phospho, phosphono, phosphoranyl, phosphoroso, siloxy, silyl,
sulfeno, sulfino, sulfo, sulfonyl, and thio. More preferably,
R.sup.1.beta., R.sup.2.beta., R.sup.3.beta., R.sup.4.beta.,
R.sup.5.beta., R.sup.8.beta., R.sup.9, R.sup.10, R.sup.11.beta.,
and R.sup.13.beta. are independently selected from the group
consisting of hydrogen and hydrocarbyl comprising from 1 to 6
carbon atoms. Most preferably, R.sup.1.beta., R.sup.2.beta.,
R.sup.3.beta., R.sup.4.beta., R.sup.5.beta., R.sup.8.beta.,
R.sup.9, R.sup.10, R.sup.11.beta., and R.sup.13.beta. are
hydrogen.
[0135] R.sup.3.alpha. preferably (a) comprises from 1 to 20 carbon
atoms (more preferably from 1 to 6 carbon atoms), and is selected
from the group consisting of hydrocarbyl and substituted
hydrocarbyl; or (b) does not comprise a carbon atom and is selected
from the group consisting of amino, halogen, hydrogen, imino,
nitro, nitroso, oximido, oxo, oxy, phosphinidene, phosphino,
phosphinyl, phosphinylidene, phospho, phosphono, phosphoranyl,
phosphoranylidene, phosphoroso, siloxy, silyl, silylene, sulfeno,
sulfinyl, sulfino, sulfo, sulfonyl, thio, and thioxo. In a more
preferred embodiment, R.sup.3.alpha. is hydrogen. In another more
preferred embodiment, R.sup.3.alpha. is selected from the group
consisting of glycosidyl, acetylated glycosidyl, and malonylated
glycosidyl. In an additional more preferred embodiment,
R.sup.3.alpha. is --OC(O)(R.sup.114), wherein R.sup.114 is benzyl
or --N(CH.sub.2CH.sub.2Cl).sub.2. In yet another more preferred
embodiment, R.sup.3.alpha. comprises (a) no greater than 20 carbon
atoms (more preferably, no greater than 6 carbon atoms); and (b) a
moiety selected from the group consisting of amino, halogen,
hydrogen, imino, oximido, oxo, oxy, phosphinidene, phosphino,
phosphinyl, phosphinylidene, phosphono, phosphoranyl,
phosphoranylidene, siloxy, silyl, silylene, sulfeno, sulfino,
sulfo, and thio. In an even more preferred embodiment,
R.sup.3.alpha. comprises (a) no greater than 20 carbon atoms (more
preferably, no greater than 6 carbon atoms); and (b) a moiety
selected from the group consisting of amino, imino, oximido, oxy,
phosphinidene, phosphino, phosphinyl, phosphinylidene, phosphono,
phosphoranyl, phosphoranylidene, siloxy, silyl, silylene, sulfeno,
sulfino, sulfo, and thio. In a still even more preferred
embodiment, R.sup.3.alpha. comprises a polarizable hydrogen atom,
and is, for example, --C(O)(OH), --NH.sub.2, .dbd.NH, .dbd.N(OH),
--OH, .dbd.PH, --PH.sub.2, --P(O)(H)(H), .dbd.P(O)(H),
--P(O)(OH)(OH), --PH.sub.4, .dbd.PH.sub.3, .dbd.SiH.sub.2, --S(OH),
--S(O)(OH), S(O)(O)(OH), or --SH. In an alternative even more
preferred embodiment, R.sup.3.alpha. is --OR.sup.115 or
--OC(O)R.sup.116, wherein R.sup.115 and R.sup.116 are hydrogen,
halogen, or hydrocarbyl comprising from 1 to 19 carbon atoms
(particularly 1 to 5 carbon atoms, and more particularly from 1 to
2 carbon atoms). Most preferably, R.sup.3.alpha. is --OH.
[0136] R.sup.4.alpha., R.sup.5.alpha., and R.sup.11.alpha.
preferably are independently a moiety which (a) comprises from 1 to
20 carbon atoms (more preferably from 1 to 6 carbon atoms), and is
selected from the group consisting of hydrocarbyl and substituted
hydrocarbyl; or (b) does not comprise a carbon atom and is selected
from the group consisting of amino, halogen, hydrogen, imino,
nitro, nitroso, oximido, oxo, oxy, phosphinidene, phosphino,
phosphinyl, phosphinylidene, phospho, phosphono, phosphoranyl,
phosphoranylidene, phosphoroso, siloxy, silyl, silylene, sulfeno,
sulfinyl, sulfino, sulfo, sulfonyl, thio, and thioxo. More
preferably, R.sup.4.alpha., R.sup.5.alpha., and R.sup.11.alpha. are
independently selected from the group consisting of hydrogen and
hydrocarbyl comprising from 1 to 6 carbon atoms. Most preferably,
R.sup.4.alpha., R.sup.5.alpha., and R.sup.11.alpha. are
hydrogen.
[0137] R.sup.8.alpha., R.sup.12.alpha., and R.sup.14.alpha.
preferably are independently a moiety which (a) comprises from 1 to
20 carbon atoms and is selected from the group consisting of
hydrocarbyl and substituted hydrocarbyl; or (b) does not comprise a
carbon atom and is selected from the group consisting of amino,
halogen, hydrogen, imino, nitro, nitroso, oximido, oxo, oxy,
phosphinidene, phosphino, phosphinyl, phosphinylidene, phospho,
phosphono, phosphoranyl, phosphoranylidene, phosphoroso, siloxy,
silyl, silylene, sulfeno, sulfinyl, sulfino, sulfo, sulfonyl, thio,
and thioxo. More preferably, R.sup.8.alpha., R.sup.12.alpha., and
R.sup.14.alpha. are independently selected from the group
consisting of hydrogen and hydrocarbyl comprising from 1 to 6
carbon atoms (particularly from 1 to 2 carbon atoms).
[0138] R.sup.13.alpha. preferably comprises (a) no greater than 20
carbon atoms (more preferably no greater than 6 carbon atoms); and
(b) a moiety selected from the group consisting of amino, imino,
oximido, oxy, phosphinidene, phosphino, phosphinyl,
phosphinylidene, phosphono, phosphoranyl, phosphoranylidene,
siloxy, silyl, silylene, sulfeno, sulfino, sulfo, and thio. More
preferably, R.sup.13.alpha. comprises a polarizable hydrogen atom
and is, for example, --C(O)(OH), --NH.sub.2, .dbd.NH, .dbd.N(OH),
--OH, .dbd.PH, --PH.sub.2, --P(O)(H)(H), .dbd.P(O)(H),
--P(O)(OH)(OH), --PH.sub.4, .dbd.PH.sub.3, .dbd.SiH.sub.2, --S(OH),
--S(O)(OH), S(O)(O)(OH), or --SH. In an alternative more preferred
embodiment, R.sup.13.alpha. is --C(O)(OR.sup.117), wherein
R.sup.117 is hydrogen, halogen, or hydrocarbyl comprising from 1 to
5 carbon atoms. Most preferably, R.sup.13.alpha. is --C(O)(OH)
(i.e., the compound is an estrogenic carboxylic acid).
[0139] R.sup.12.beta. and R.sup.14.beta. preferably are
independently a moiety which (a) comprises from 1 to 20 carbon
atoms and is selected from the group consisting of hydrocarbyl and
substituted hydrocarbyl; or (b) does not comprise a carbon atom and
is selected from the group consisting of amino, halogen, hydrogen,
nitro, nitroso, oxy, phosphino, phosphinyl, phospho, phosphono,
phosphoranyl, phosphoroso, siloxy, silyl, sulfeno, sulfino, sulfo,
sulfonyl, and thio. More preferably, R.sup.12.beta. and
R.sup.14.beta. are independently selected from the group consisting
of hydrogen and hydrocarbyl comprising from 1 to 6 carbon atoms
(particularly from 1 to 2 carbon atoms).
[0140] When the compound has the structure of formula (XIII) or is
a pharmaceutically acceptable salt thereof, it is particularly
preferred for the compound to have formula (XIV) or be a
pharmaceutically acceptable thereof: 19
[0141] Here, X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.8,
X.sup.9, X.sup.10, X.sup.11, X.sup.12, X.sup.13, X.sup.14,
R.sup.8.alpha., R.sup.8.beta., R.sup.9, R.sup.11.alpha.,
R.sup.11.beta., R.sup.12.alpha., R.sup.12.beta., R.sup.13.alpha.,
R.sup.13.beta., R.sup.14.alpha., and R.sup.14.GAMMA. preferably are
as defined as in formula (XIII).
[0142] The dashed lines are optional .pi. bonds.
[0143] R.sup.8.beta., R.sup.9, R.sup.12.beta., R.sup.13.beta.,
and/or R.sup.14.beta. are present only when X.sup.8, X.sup.9,
X.sup.11, X.sup.12, X.sup.13, and/or X.sup.14, respectively, are
saturated.
[0144] R.sup.1.alpha. and R.sup.2.alpha. preferably are
independently a moiety which (a) comprises from 1 to 20 carbon
atoms (more preferably from 1 to 6 carbon atoms), and is selected
from the group consisting of hydrocarbyl and substituted
hydrocarbyl; or (b) does not comprise a carbon atom and is selected
from the group consisting of amino, halogen, hydrogen, nitro,
nitroso, oxy, phosphino, phosphinyl, phospho, phosphono,
phosphoranyl, phosphoroso, siloxy, silyl, sulfeno, sulfino, sulfo,
sulfonyl, and thio. More preferably, R.sup.1.alpha. and
R.sup.2.alpha. are independently selected from the group consisting
of hydrogen; hydrocarbyl comprising from 1 to 6 carbon atoms; and
--OR.sup.118, wherein R.sup.118 is hydrogen or hydrocarbyl
containing from 1 to 6 carbon atoms, and particularly wherein
R.sup.118 is hydrogen or methyl. Most preferably, R.sup.1.alpha.
and R.sup.2.alpha. are hydrogen.
[0145] R.sup.3.alpha. preferably (a) comprises from 1 to 20 carbon
atoms (more preferably from 1 to 6 carbon atoms), and is selected
from the group consisting of hydrocarbyl and substituted
hydrocarbyl; or (b) does not comprise a carbon atom and is selected
from the group consisting of amino, halogen, hydrogen, nitro,
nitroso, oxy, phosphino, phosphinyl, phospho, phosphono,
phosphoranyl, phosphoroso, siloxy, silyl, sulfeno, sulfino, sulfo,
sulfonyl, and thio. In a more preferred embodiment, R.sup.3.alpha.
is hydrogen. In another more preferred embodiment, R.sup.3.alpha.
is selected from the group consisting of glycosidyl, acetylated
glycosidyl, and malonylated glycosidyl. In an additional more
preferred embodiment, R.sup.3.alpha. is --OC(O)(R.sup.119), wherein
R.sup.119 is benzyl or --N(CH.sub.2CH.sub.2Cl).sub.2. In yet
another more preferred embodiment, R.sup.3.alpha. comprises (a) no
greater than 20 carbon atoms (more preferably, no greater than 6
carbon atoms); and (b) a moiety selected from the group consisting
of amino, imino, oximido, oxy, phosphinidene, phosphino,
phosphinyl, phosphinylidene, phosphono, phosphoranyl,
phosphoranylidene, siloxy, silyl, silylene, sulfeno, sulfino,
sulfo, and thio. In an even more preferred embodiment,
R.sup.3.alpha. comprises a polarizable hydrogen atom and is, for
example, --C(O)(OH), --NH.sub.2, .dbd.NH, .dbd.N(OH), --OH,
.dbd.PH, --PH.sub.2, --P(O)(H)(H), .dbd.P(O)(H), --P(O)(OH)(OH),
--PH.sub.4, .dbd.PH.sub.3, .dbd.SiH.sub.2, --S(OH), --S(O)(OH),
S(O)(O)(OH), or --SH. In an alternative even more preferred
embodiment, R.sup.3.alpha. is --OR.sup.120 or --OC(O)R.sup.121,
wherein R.sup.120 and R.sup.121 are hydrogen, halogen, or
hydrocarbyl comprising from 1 to 19 carbon atoms (particularly 1 to
5 carbon atoms, and more particularly 1 to 2 carbon atoms). Most
preferably, R.sup.3.alpha. is --OH.
[0146] R.sup.4.alpha. and R.sup.5.alpha. preferably are
independently a moiety which (a) comprises from 1 to 20 carbon
atoms (more preferably from 1 to 6 atoms), and is selected from the
group consisting of hydrocarbyl and substituted hydrocarbyl; or (b)
does not comprise a carbon atom and is selected from the group
consisting of amino, halogen, hydrogen, nitro, nitroso, oxy,
phosphino, phosphinyl, phospho, phosphono, phosphoranyl,
phosphoroso, siloxy, silyl, sulfeno, sulfino, sulfo, sulfonyl, and
thio. More preferably, R.sup.4.alpha. and R.sup.5.alpha. are
independently selected from the group consisting of hydrogen and
hydrocarbyl comprising from 1 to 6 carbon atoms. Most preferably,
R.sup.4.alpha. and R.sup.5.alpha. are hydrogen.
[0147] Examples of preferred compounds having the structure of
formula (XIII) include the following estrogenic carboxylic acids
(and pharmacuetically acceptable salts thereof):
[0148] 1.
1-(p-hydroxyphenyl)-6-ethyl-5-methylcyclohexene-4-carboxylic acid:
20
[0149] 2.
1-(p-hydroxyphenyl)-2-ethyl-3-methylcyclohexene-4-carboxylic acid:
21
[0150] 3.
1-(p-hydroxyphenyl)-2-ethyl-3,5,5-trimethylcyclohexene-4-carboxy-
lic acid: 22
[0151] 4. 4-(p-hydroxyphenyl)-2,2,6,6-tetramethylcyclohexane
carboxylic acid: 23
[0152] 5. 1-phenyl-2-ethyl-3-methylcyclohexene-4-carboxylic acid:
24
[0153] 6. 1-phenyl-5,6-dimethylcyclohexene-4-carboxylic acid:
25
[0154] For a review of the structures of estrogenic doisynolic-type
acids, and methods for preparing these compounds, the reader is
referred to the review of Meyers et al., "Doisynolic-Type
Acids-Uterotropically Potent Estrogens Which Compete Poorly With
Estradiol for Cytosolic Estradiol Receptors, J. Steroid Biochem.
31(4A):393-404 (1988), and the references cited therein.
[0155] B. Pharmaceutical Compositions
[0156] The compounds of the present invention can be formulated as
pharmaceutical compositions. Such compositions can be administered
orally, parenterally, by inhalation spray, rectally, intradermally,
transdermally, or topically in dosage unit formulations containing
conventional nontoxic pharmaceutically acceptable carriers,
adjuvants, and vehicles as desired. Topical administration may also
involve the use of transdermal administration such as transdermal
patches or iontophoresis devices. The term parenteral as used
herein includes subcutaneous, intravenous, intramuscular, or
intrastemal injection, or infusion techniques. Formulation of drugs
is discussed in, for example, Hoover, John E., Remington's
Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania
(1975), and Liberman, H. A. and Lachman, L., Eds., Pharmaceutical
Dosage Forms, Marcel Decker, New York, N.Y. (1980).
[0157] Injectable preparations, for example, sterile injectable
aqueous or oleaginous suspensions, can be formulated according to
the known art using suitable dispersing or wetting agents and
suspending agents. The sterile injectable preparation may also be a
sterile injectable solution or suspension in a nontoxic
parenterally acceptable diluent or solvent, for example, as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution, and
isotonic sodium chloride solution. In addition, sterile, fixed oils
are conventionally employed as a solvent or suspending medium. For
this purpose, any bland fixed oil may be employed, including
synthetic mono- or diglycerides. In addition, fatty acids such as
oleic acid are useful in the preparation of injectables. Dimethyl
acetamide, surfactants including ionic and non-ionic detergents,
and polyethylene glycols can be used. Mixtures of solvents and
wetting agents such as those discussed above are also useful.
[0158] Suppositories for rectal administration of the compounds
discussed herein can be prepared by mixing the active agent with a
suitable non-irritating excipient such as cocoa butter, synthetic
mono-, di-, or triglycerides, fatty acids, or polyethylene glycols
which are solid at ordinary temperatures but liquid at the rectal
temperature, and which will therefore melt in the rectum and
release the drug.
[0159] Solid dosage forms for oral administration may include
capsules, tablets, pills, powders, and granules. In such solid
dosage forms, the compounds of this invention are ordinarily
combined with one or more adjuvants appropriate to the indicated
route of administration. If administered per os, the compounds can
be admixed with lactose, sucrose, starch powder, cellulose esters
of alkanoic acids, cellulose alkyl esters, talc, stearic acid,
magnesium stearate, magnesium oxide, sodium and calcium salts of
phosphoric and sulfuric acids, gelatin, acacia gum, sodium
alginate, polyvinylpyrrolidone, and/or polyvinyl alcohol, and then
tableted or encapsulated for convenient administration. Such
capsules or tablets can contain a controlled-release formulation as
can be provided in a dispersion of active compound in
hydroxypropylmethyl cellulose. In the case of capsules, tablets,
and pills, the dosage forms can also comprise buffering agents such
as sodium citrate, or magnesium or calcium carbonate or
bicarbonate. Tablets and pills can additionally be prepared with
enteric coatings.
[0160] For therapeutic purposes, formulations for parenteral
administration can be in the form of aqueous or non-aqueous
isotonic sterile injection solutions or suspensions. These
solutions and suspensions can be prepared from sterile powders or
granules having one or more of the carriers or diluents mentioned
for use in the formulations for oral administration. The compounds
can be dissolved in water, polyethylene glycol, propylene glycol,
ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl
alcohol, sodium chloride, and/or various buffers. Other adjuvants
and modes of administration are well and widely known in the
pharmaceutical art.
[0161] Liquid dosage forms for oral administration can include
pharmaceutically acceptable emulsions, solutions, suspensions,
syrups, and elixirs containing inert diluents commonly used in the
art, such as water. Such compositions can also comprise adjuvants,
such as wetting agents, emulsifying and suspending agents, and
sweetening, flavoring, and perfuming agents.
[0162] The amount of active ingredient that can be combined with
the carrier materials to produce a single dosage form will vary
depending upon the patient and the particular mode of
administration.
[0163] The mode of administration is partially dependent upon the
chemical form of the estrogenic carboxylic acids. The phenolic and
carboxylic salts (e.g., sodium, potassium, calcium, etc.) are more
water soluble than the parent phenolic carboxylic acids, and can be
administered orally or in aqueous solution. The estrogenic
carboxylic acids themselves and their esters and related
derivatives have lower water solubility and are probably best
administered subcutaneously or transdermally in an oily or
penetrating vehicle.
[0164] In the form of their 3-methyl ethers, (.+-.)-Z-BDDA
("Fenocylin", Ciba-Geigy) and allenolic acid ("Vallestril", G. D.
Searle and Co.) have been cleared for clinical use. However,
Segaloff ((1949) in Recent Progress in Hormone Research, Vol. IV,
G. Pincus, Ed., Academic Press, New York, pp. 85-111) discounted
the clinical activity of Fenocylin in women. See Meyers C Y, Kolb V
M, Gass G H, Rao B R, Roos C F, Dandliker W B: "Doisynolic-Type
Acids--Uterotropically Potent Estrogens which Compete Poorly with
Estradiol for Cytosolic Estradiol Receptors. J Steroid Biochem
31:393-404 (1988); and Soto A M, Meyers C Y, Sonnenschein C: "How
Many Rings Can be Cleaved from a Steroidal Estrogen While
Preserving its Estrogenic Activity?"The Endocrine Society, 70th
Annual Meeting, Abstract (1988); and the foregoing discussion.
[0165] Certain of the pharmaceutical compounds of this invention
which are administered in accordance with the methods of the
invention can serve as prodrugs to other compounds of this
invention. Prodrugs are drugs that can be chemically converted in
vivo or in vitro by biological systems into an active derivative or
derivatives. Prodrugs are administered in essentially the same
fashion as the other pharmaceutical compounds of the invention.
Non-limiting examples include non-hydroxylated
phenylcyclohexenecarboxylic acids of this invention that are
hydroxylated in vivo.
[0166] It should be noted that the present invention encompasses
the use of estrogenic carboxylic acids as disclosed herein
formulated alone, and in various combinations with one another.
Single estrogenic carboxylic acids, or combinations of estrogenic
carboxylic acids, can also be formulated in combination with other
estrogens coventionally used in the art.
[0167] C. Dosages
[0168] Depending upon the particular pharmaceutical application,
the estrogenically active compounds of the present invention can be
administered daily to humans or animals in a number of different
dosages. For example, as suggested by the results disclosed in
Example 2, below, the dosage can be an amount in the range of from
about 0.1 .mu.g/kg/day to about 100 mg/kg/day, preferably from
about 0.5 .mu.g/kg/day to about 75 mg/kg/day, more preferably from
about 1 .mu.g/kg/day to about 50 mg/kg/day, even more preferably
from about 1 .mu.g/kg/day to about 25 mg/kg/day, and still more
preferably from about 1 .mu.g/kg/day to about 20 mg/kg/day. As
suggested by the results disclosed in Example 4, below, dosages for
use in treating prostatic (and other) disorders can be an amount in
the range of from about 10 .mu.g/kg/day to about 10 mg/kg/day,
preferably from about 10 .mu.g/kg/day to about 5 mg/kg/day, more
preferably from about 10 .mu.g/kg/day to about 2.5 mg/kg/day, and
even more preferably from about 10 .mu.g/kg/day to about 1
mg/kg/day. In further embodiments, the lower value of these dosage
ranges can be as low as about 1 .mu.g/kg/day.
[0169] The doses described above can be administered to a patient
in a single dose or in proportionate multiple subdoses, for example
two subdoses daily. In the case of proportionate multiple subdoses,
dosage unit compositions can contain such amounts of submultiples
thereof to make up the daily dose. Multiple doses per day can also
increase the total daily dose should this be desired by the person
prescribing the drug.
[0170] D. Treatment Regimen
[0171] The present invention provides methods for treating or
preventing a variety of symptoms, conditions, and diseases that
would benefit from estrogen therapy using the compounds disclosed
herein. In this context, "treating" refers to ameliorating,
suppressing, or eradicating these symptoms, conditions, and
diseases. The regimen for treating a patient suffering from a
symptom, condition, or disease that would benefit from estrogen
therapy, or preventing the same, with the compounds and/or
compositions of the present invention is selected in accordance
with a variety of factors, including the age, weight, sex, diet,
and medical condition of the patient, the severity of the
infection, the route of administration, pharmacological
considerations such as the activity, efficacy, pharmacokinetic, and
toxicology profiles of the particular compounds employed, and
whether a drug delivery system is utilized. It should be noted that
the methods disclosed herein are applicable in both human and
veterinary medicine. Treatment of domestic pets, such as cats and
dogs, is contemplated in the present invention.
[0172] Administration of individual estrogenic carboxylic acids,
combinations thereof, or such individual estrogenic carboxylic
acids or combinations thereof in further combination with estrogens
conventionally used in the art, should generally be continued over
a period of several weeks to several months or years until symptoms
reach acceptable levels, or have been eliminated entirely,
indicating that the condition has been controlled or eradicated. As
noted above, patients undergoing treatment with the drugs disclosed
herein can be routinely monitored by measuring appropriate physical
and physiological parameters to determine the effectiveness of
therapy.
[0173] Continuous analysis of the data obtained by these methods
permits modification of the treatment regimen during therapy so
that optimal amounts of each compound are administered, and so that
the duration of treatment can be determined as well. Thus, the
treatment regimen/dosing schedule can be rationally modified over
the course of therapy so that the lowest amount of each estrogenic
carboxylic acid used alone or in combination which together exhibit
satisfactory therapeutic effectiveness are administered, and so
that administration of such compounds is continued only so long as
is necessary to successfully treat the indicated condition.
[0174] In order to monitor the effect and progress of treatment,
conventional assays can be used wherever appropriate. For example,
the standard immunoassays for testosterone and prostate specific
antigen (PSA) can be used in the case of prostate cancer.
Significant decreases in either testosterone or PSA indicate the
utility of the present compounds as therapeutic agents. When such
assays are lacking or where effects are expected to be very slow,
more subjective parameters can be employed. These are considered
individually in the following examples discussing each disease.
[0175] The chronic effects of the Z-BDDA compounds as compared to
those of E2 were studied in rats as a model mammalian system. In
addition to anlysis of changes in body weight, additional metabolic
and endocrine studies were performed, including monitoring food
intake and metabolic and reproductive parameters in male and female
rats. Because so little has been reported on the comparative
effects of the Z-BDDA enantiomers, the (+)-, (-)-, and (.+-.)-forms
were prepared and investigated. Anner G, Miescher K:
Hydrierungs-Und Umlagerungs-Reaktion in der Doisynolsure-Reihe.
Oestrogene Carbonsuren XII. Helv. Chim. Acta 29 (1946) 1889-1895;
Die totalsyntheses von racemischen doisynolsuren XXI. ber
oestrogene carbonsueren. ibid 30:1422-1432 (1947); Tschopp E:
"Wirksamkeit, organconzentration und ausscheidung der
7-methyl-bisdehydro-doisynolsure." Helv Physiol Pharmacol Acta
4:401-410 (1946); Tschopp E: "Die oestrogene wirkung der
bisdehydrodoisynolsure und ihre derivate." Helv Physiol Pharmacol
Acta 4:271-284 (1946); Rometsch R, Miescher K: "Die spaltung des
racemates der n-bisdehydro-doisynolsure. ber ostrogene carbonsuren
X." Helv Chim Acta 29:1231-1235 (1946); and Terenius L:
"Differential Inhibition In Vitro of 17.beta.-Estradiol Binding in
the Mouse Uterus and Vagina by Optical Antipodes of Estrogen."
Molec Pharmac 4:301-310 (1968).
[0176] Definitions
[0177] The term "acyl" means the group having the formula
--C(O)(R), wherein R is hydrocarbyl. The term "substituted acyl"
means the group having the formula --C(O)(R), wherein R is, for
example, substituted hydrocarbyl.
[0178] The term "alkanoyl halide" means the group having the
formula --C(O)(R), wherein R is halogen.
[0179] The term "alkenyl" means a straight or branched hydrocarbyl
comprising at least one carbon-carbon double bond, and includes,
for example, ethenyl, propenyl, iso-propenyl, butenyl, isobutenyl,
hexenyl, and the like.
[0180] The term "alkyl" means a saturated straight or branched
chain hydrocarbyl (i.e., no double or triple carbon-carbon bonds),
and includes, for example, methyl, ethyl, n-propyl, isopropyl,
n-butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, hexyl, and the
like.
[0181] The term "alkynyl" means a straight or branched hydrocarbyl
comprising at least one triple carbon-carbon bond, and includes,
for example, ethynyl, propynyl, butynyl, isobutynyl, hexynyl, and
the like.
[0182] The term "amide" means the group having the formula
--C(O)(N(R.sup.a)(R.sup.b)), wherein R.sup.a and R.sup.b are
independently, for example, hydrogen, hydrocarbyl, or substituted
hydrocarbyl.
[0183] The term "amino" means the group having the formula
--N(R.sup.a)(R.sup.b), wherein R.sup.a and R.sup.b are
independently, for example, hydrogen, hydrocarbyl, or substituted
hydrocarbyl.
[0184] The term "carboxyl" means the group having the formula
--C(O)(OR), wherein R is, for example, hydrogen, hydrocarbyl, or
substituted hydrocarbyl.
[0185] The term "formyl" means the group having the formula
--C(H)(O).
[0186] The term "halogen" includes F, Cl, Br, and I.
[0187] The term "heterocyclyl" means a chain of 3 or more atoms
(typically 5 or 6 atoms) forming a ring (or multiple rings),
wherein at least one of the atoms forming the ring is an atom which
is not a carbon atom or hydrogen atom (e.g., sulfur, nitrogen, or
oxygen). The heterocyclyl may comprise all single bonds between the
atoms forming the ring, or, alternatively, may comprise one or more
double bonds between such atoms. Heterocyclyls include, for
example, furyl, thienyl, pyridinyl, morpholinyl, and the like. In
addition to being bound to the other atoms forming the ring, the
atoms forming the ring of the heterocyclyl may also be bound to
hydrogen or to another group, such as, for example: (a) a group
which is selected from the group consisting of hydrocarbyl or a
substituted hydrocarbyl (e.g., another heterocyclyl); or (b) a
group which does not comprise a carbon atom and is selected from
the group consisting of an amino, halogen, hydrogen, imino, nitro,
nitroso, oximido, oxo, oxy, phosphinidene, phosphino, phosphinyl,
phosphinylidene, phospho, phosphono, phosphoranyl,
phosphoranylidene, phosphoroso, siloxy, silyl, silylene, sulfeno,
sulfino, sulfinyl, sulfo, sulfonyl, thio, and thioxo.
[0188] The term "hydrocarbyl" means a group consisting exclusively
of carbon and hydrogen atoms. Such a group may be straight,
branched, cyclic (or multi-cyclic), or a combination thereof. It
may also be saturated (i.e., comprise no carbon-carbon double or
triple bonds) or unsaturated (i.e., comprise at least one
carbon-carbon double or triple bond). Hydrocarbyls include, for
example, alkyl, alkenyl, alkynyl, aryl, alkaryl, alkenaryl, and
alkynaryl. The term "substituted hydrocarbyl" means a hydrocarbyl,
wherein at least one hydrogen atom has been substituted with (a) an
atom which is not a hydrogen or carbon atom (i.e., a heteroatom),
or (b) a group of atoms comprising at least one heteroatom. A
"heteroatom" may be, for example, a boron, halogen, nitrogen,
oxygen, phosphorous, silicon, or sulfur atom. Substituted
hydrocarbyls include hydrocarbyls wherein one or more hydrogen
atoms have been substituted with, for example, amino, halogen,
heterocyclyl, imino, nitro, nitroso, oximido, oxo, oxy,
phosphinidene, phosphino, phosphinyl, phosphinylidene, phospho,
phosphono, phosphoranyl, phosphoranylidene, phosphoroso, siloxy,
silyl, silylene, sulfeno, sulfinyl, sulfino, sulfo, sulfonyl, thio,
or thioxo. Examples of substituted hydrocarbyls include acyl (e.g.,
acetyl and benzoyl) and substituted acyl, alkanoyl halide, amide,
formyl, nitrile, carboxyl, oxycarbonyl, alkoxy, amino substituted
with hydrocarbyl (i.e., N(R.sup.a)(R.sup.b), wherein R.sup.a and
R.sup.b are hydrocarbyl), phosphono substituted with hydrocarbyl
(i.e., a phosphono ester, --P(O(OR.sup.a)(OR.sup.b), wherein
R.sup.a and R.sup.b are hydrocarbyl), and sulfo substituted with
hydrocarbyl (i.e., a sulfo ester, --S(O)(O)(OR), wherein R is
hydrocarbyl).
[0189] The term "imino" means the group having the formula.dbd.NR,
wherein R is, for example, hydrogen, hydrocarbyl, or substituted
hydrocarbyl.
[0190] The term "nitrile" means the group having the formula
--CN.
[0191] The term "nitro" means the group having the formula
--NO.sub.2.
[0192] The term "nitroso" means the group having the formula
--NO.
[0193] The term "non-hydrocarbyl group" means a group that
comprises no carbon atoms.
[0194] The term "oximido" means the group having the formula
.dbd.N(OR), wherein R is, for example, hydrogen, hydrocarbyl, or
substituted hydrocarbyl.
[0195] The term "oxo" means the oxygen group (i.e., .dbd.O) of a
carbonyl group.
[0196] The term "oxy" means the group having the formula --OR,
wherein R is, for example, hydrogen (i.e., --OR is hydroxy),
hydrocarbyl, or substituted hydrocarbyl.
[0197] The term "oxycarbonyl" means the group having the formula
--OC(O)(R), wherein R is, for example, hydrocarbyl or substituted
hydrocarbyl.
[0198] The term "pharmaceutically acceptable salt" embraces salts
commonly used to form alkali metal salts and to form addition salts
of free acids or free bases. The nature of the salt is not
critical, provided that it is pharmaceutically-acceptable. Suitable
pharmaceutically-acceptable acid addition salts of the therapeutic
compounds discussed herein may be prepared from an inorganic acid
or from an organic acid. Examples of such inorganic acids are
hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric
and phosphoric acid. Appropriate organic acids may be selected from
aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclyl,
carboxylic and sulfonic classes of organic acids, example of which
are formic, acetic, propionic, succinic, glycolic, gluconic,
lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic,
fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic,
mesylic, stearic, salicylic, p-hydroxybenzoic, phenylacetic,
mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic,
benzenesulfonic, pantothenic, toluenesulfonic,
2-hydroxyethanesulfonic, sulfanilic, cyclohexylaminosulfonic,
algenic, b-hydroxybutyric, galactaric and galacturonic acid.
Suitable pharmaceutically-acceptable base addition salts of the
therapeutic compounds discussed herein include metallic salts and
organic salts. More preferred metallic salts include, but are not
limited to, appropriate alkali metal (group Ia) salts, alkaline
earth metal (group IIa) salts, and other physiological acceptable
metals. Such salts can be made from aluminum, calcium, lithium,
magnesium, potassium, sodium and zinc. Preferred organic salts can
be made from tertiary amines and quaternary ammonium salts,
including in part, tromethamine, diethylamine,
N,N'-dibenzylethylenediamine, chloroprocaine, choline,
diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and
procaine. All of these salts may be prepared by conventional means
from the corresponding therapeutic compounds discussed herein by
reacting, for example, the appropriate acid or base with the
compounds.
[0199] The term "phosphinidene" means the group having the formula
.dbd.PR, wherein R is, for example, hydrogen, hydrocarbyl, or
substituted hydrocarbyl.
[0200] The term "phosphino" means the group having the formula
--P(R.sup.a)(R.sup.b), wherein R.sup.a and R.sup.b are
independently, for example, hydrogen, hydrocarbyl, or substituted
hydrocarbyl.
[0201] The term "phosphinyl" means the group having the formula
--P(O)(R.sup.a)(R.sup.b), wherein R.sup.a and R.sup.b are
independently, for example, hydrogen, hydrocarbyl, or substituted
hydrocarbyl.
[0202] The term "phosphinylidene" means the group having the
formula .dbd.P(O)(R), wherein R is, for example, hydrogen,
hydrocarbyl, or substituted hydrocarbyl.
[0203] The term "phospho" means the group having the formula
--PO.sub.2.
[0204] The term "phosphono" means the group having the formula
--P(O)(OR.sup.a)(OR.sup.b), wherein R.sup.a and R.sup.b are
independently, for example, hydrogen, hydrocarbyl, or substituted
hydrocarbyl.
[0205] The term "phosphoranyl" means the group having the formula
--P(R.sup.a)(R.sup.b)(RC)(Rd), wherein R.sup.a, R.sup.b, R.sup.c,
and R.sup.d are independently, for example, hydrogen, hydrocarbyl,
or substituted hydrocarbyl.
[0206] The term "phosphoranylidene" means the group having the
formula .dbd.P(R.sup.a)(R.sup.b)(R.sup.c), wherein R.sup.a,
R.sup.b, and R.sup.c are independently, for example, hydrogen,
hydrocarbyl, or substituted hydrocarbyl.
[0207] The term "phosphoroso" means the group having the formula
--PO.
[0208] The term "siloxy" means the group having the formula
--OSi(R.sup.a)(R.sup.b)(R.sup.c), wherein R.sup.a, R.sup.b, and
R.sup.c are independently, for example, hydrogen, hydrocarbyl, or
substituted hydrocarbyl.
[0209] The term "silyl" means the group having the formula
--Si(R.sup.a)(R.sup.b)(c), wherein R.sup.a, R.sup.b, and R.sup.c
are independently, for example, hydrogen, hydrocarbyl, or
substituted hydrocarbyl.
[0210] The term "silylene" means the group having the formula
.dbd.Si(R.sup.a)(R.sup.b), wherein R.sup.a and R.sup.b are
independently, for example, hydrogen, hydrocarbyl, or substituted
hydrocarbyl.
[0211] The term "unsubstituted silylene" means the group having the
formula .dbd.SiH.sub.2.
[0212] The term "sulfeno" means the group having the formula
--S(OR), wherein R is, for example, hydrocarbyl or substituted
hydrocarbyl.
[0213] The term "sulfino" means the group having the formula
--S(O)(OH). The term "substituted sulfino" means the group having
the formula --S(O)(OR), wherein R is, for example, hydrocarbyl or
substituted hydrocarbyl.
[0214] The term "sulfinyl" means the group having the formula
.dbd.SO.
[0215] The term "sulfo" means the group having the formula
--S(O)(O)(OR), wherein R is, for example, hydrogen, hydrocarbyl, or
substituted hydrocarbyl.
[0216] The term "sulfonyl" means the group having the formula
--S(O)(O)(R), wherein R is, for example, halogen, hydrocarbyl,
substituted hydrocarbyl, or amine.
[0217] The term "thio" means the group having the formula --SR,
wherein R is, for example, hydrogen, hydrocarbyl, or substituted
hydrocarbyl.
[0218] The term "thioxo" means the group having the formula
.dbd.S.
EXAMPLES
[0219] The following non-limiting examples illustrate various
aspects of the present invention.
Example 1
Preparation of (.+-.-Z-bis dehydrodoisynolic acid
((.+-.)-Z-BDDA))
[0220] (.+-.)-Z-bisdehydrodoisynolic acid ((.+-.)-Z-BDDA)) is
prepared by refluxing a solution of Fenocylin in concentrated
HBr-HOAc for 2.5 hr. After recrystallization, the melting point is
204-205.5.degree. C. Elemental analysis, acid-base titration, and
NMR can be used to confirm the identify of the product.
[0221] Alternatively, potassium hydroxide fusion of equilenin
yields a mixture of acids from which the
(-)-Z-bis-dehydrodoisynolic acid can be isolated (K. Miescher,
Chem. Rev. 43:367-384 (1948)). One or more of the allenolic acids
can be prepared by literature methods cited in Miescher. The other
non-steroidal estrogenic compounds of the present invention, i.e.,
the hydroxyphenylcyclohexane-and -cyclo-hexene-, and
hydroxytetrahydroanthracenecarboxylic acids disclosed herein, can
be synthesized by methods disclosed in references discussed in
Meyers et al., J. Steroid Biochem. 31(4A):393-404 (1988).
Example 2
Comparative Effects of (-)-, (+)-, and
(.+-.)-Z-bisdehydrodoisynolic Acids and Estradiol on Body Weight,
Food Intake, Metabolic, and Reproductive Parameters in Male and
Female Rats
[0222] A study was designed to investigate the chronic effects of
the Z-BDDA compounds vs. E2 in rats. In addition to analysis of
changes in body weight, additional metabolic and endocrine studies
were performed, including monitoring food intake and metabolic and
reproductive parameters, in male and female rats. Moreover, because
so little has been reported on the comparative effects of the
Z-BDDA enantiomers, the (+), (-) and (.+-.) forms were prepared and
investigated.
[0223] The compounds used in these studies were
(+)-17.beta.-estradiol (E2), (-)-Z-bisdehydrodoisynolic acid
[(-)-Z-BDDA], and (+)-Z-bisdehydrodoisynolic acid [(.+-.)-Z-BDDA].
Their structures are shown below in a, b, and c, respectively.
Racemic (.+-.)-Z-bisdehydrodois- ynolic acid [(.+-.)-Z-BDDA] is a
1:1 mixture of the (+) and the (-) enantiomers. 26
[0224] (+)-17.beta.-Estradiol (E2) was purchased from Sigma
Chemical Company (St. Louis, Mo.). (.+-.)-Z-Bisdehydrodoisynolic
acid [((.+-.)-Z-BDDA] was prepared from (.+-.)-Z-BDDA-3-OMe
("Fenocylin," from Ciba-Geigy, Inc.; m.p. 228-230.degree. C) as
described by Meyers et al. (Meyers C Y, Kolb V M, Gass G H, Rao B
R, Roos C F, Dandliker W B: "Doisynolic-Type Acids-Uterotropically
Potent Estrogens which Compete Poorly with Estradiol for Cytosolic
Estradiol Receptors. J Steroid Biochem 31:393-404 (1988); and Banz
J, Winters T A, Hou Y, Adler S, Meyers C Y: "Activities of
Non--Classical Estrogens: Effects of (-)-, (+)-, and
(.+-.)-Z-Bisdehydrodoisynolic Acids In Vitro and on Body Weight in
Male and Female Rats." The Endocrine Society, 80th Annual Meeting,
Abstract (1998)), m.p. (recrystallized from acetone-ligroin)
204-205.5.degree. C.(darkens) 204.degree. C. The slightly off-white
crystals rapidly deepen in color in solution and more slowly in
air. (+)-Z-BDDA and (-)-Z-BDDA were prepared by the resolution of
(.+-.)-Z-BDDA-3-OMe through their respective isolated and purified
L-menthyl esters according to the method reported by Rometsch and
Miescher. Rometsch R, Miescher K: "Die spaltung des racemates der
n-bisdehydro-doisynolsure. ber ostrogene carbonsuren X." Helv Chim
Acta 29:1231-1235 (1946). The crystalline (+)- and (-)-Z-BDDA so
prepared exhibited a single TLC spot, and their .sup.1H- and
.sup.13C-NMR spectra correctly identified their structure.
[0225] Experiment 1
[0226] Twenty-five male and 25 female Sprague-Dawley rats, 9-10
weeks of age, were randomly assigned to groups of five animals,
respectively, as a control group (no treatment) (C), for treatment
with vehicle only (V), estradiol (E), (.+-.)-Z-BDDA, or (+)-Z-BDDA.
Each animal in group V received a daily 0.1 cc injection of a 10%
ethanol-90% olive oil solution; the other treatments received their
respective compound as a daily 0.1 cc injection, e.g., 2.5 .mu.g of
compound/g of body weight, in a 10% ethanol-90% olive oil solution.
A temperature of 21.degree. C. and an artificial 12-h light-dark
cycle was maintained in the animal room. All animals were
maintained on standard chow in powdered form for four weeks, then
sacrificed after an overnight fast under i.p. pentobarbital
anesthesia (50 mg/kg). Animal weight and food intake were measured
weekly during the study, and the subsequent food-efficiency ratio
[FER(g of weight change/g of food intake)] was determined. During
sacrifice, blood was collected (via cardiac puncture) for
cholesterol measurements. Immediately following sacrifice the fat
pads and reproductive organs were removed and weighed.
[0227] Experiment 2
[0228] Twenty-five male and 25 female Wistar rats, 7-8 weeks of
age, were randomly assigned to groups of five animals,
respectively, for treatment with vehicle only (V), estradiol (E),
(.+-.)-Z-BDDA, (+)-Z-BDDA, or (-)-Z-BDDA. Each animal in group V
received a daily 0.1 cc injection of a 10% ethanol-90% olive oil
solution; the other treatments received their respective compound
as a daily 0.1 cc injection, e.g., 2.5 .mu.g of compound/g of body
weight, in a 10% ethanol-90% olive oil solution. A temperature of
21.degree. C. and an artificial 12-h light-dark cycle were
maintained in the animal room. All animals were maintained on
standard chow in powdered form, the males for five weeks and the
females for six weeks, then sacrificed after an overnight fast
under i.p. pentobarbital anesthesia (50 mg/kg). Animal weight and
food intake were measured weekly during the study and subsequent
FER values were determined. During sacrifice, blood was collected
(via cardiac puncture) for glucose, luteinizing hormone, prolactin
and testosterone measurements. The amounts of luteinizing hormone,
prolactin and testosterone were measured to help elucidate the
target tissue of the respective compounds in male and female rats.
Immediately following sacrifice the fat pads and reproductive
organs were removed and weighed.
[0229] Statistical Analyses
[0230] Experiments 1 and 2 employed a randomized design. All data
were analyzed by one-way analysis of variance (ANOVA,)and post-hoc
comparisons were made with Tukey pairwise comparisons test.
Significance was confirmed at p.ltoreq.0.05 (SYSTAT 7.0, SPSS INC.,
1997), and all values are reported as means.+-.standard error of
the mean.
Results
[0231] E2 and the three forms of Z-BDDA produced both similar and
distinct effects on reproductive parameters in male and female
rats. For example, the results demonstrate that E2 and (+)-and
(.+-.)-Z-BDDA behave similarly in their effect on
reproductive-organ weight: they promote an increase in uterine
weight and a decrease in testis weight, compared to the control or
vehicle alone (Table 1 and 2). Surprisingly, (-)-Z-BDDA did not
induce an increase (p<0.05) in uterine weight as observed with
(+), (.+-.)-Z-BDDA and estradiol (FIG. 1) but, like them,
(-)-Z-BDDA, compared to the vehicle alone, caused weight reduction
(p<0.05) of the testis and prostate (Tables 1 and 2).
1TABLE 1 Experiment 1. The effects of (+)-and
(.+-.)-Z-bisdehydrodoisynolic acid (Z-BDDA) and
(+)-17.beta.-estradiol on metabolic and reproductive parameters in
male and female rats on treatment for 4 weeks* Cholesterol
Uterus/testis Prostate Treatment Food intake (g) FER (food
efficiency ratio) (mg/dl) Visceral fat (g) weight (g) weight (g)
Female control 300.0 .+-. 28.6 0.079 .+-. 0.010 91.6 .+-. 12.3 2.9
.+-. 0.3 0.42 .+-. 0.03 -- vehicle.sup..dagger. 293.0 .+-. 26.1
0.040 .+-. 0.013 67.2 .+-. 12.5 1.6 .+-. 0.4.sup.1 0.66 .+-. 0.06
-- estradiol.sup..paragraph. 371.6 .+-. 17.8 0.035 .+-. 0.011 44.9
.+-. 15.8.sup.1 0.8 .+-. 0.1.sup.1 1.58 .+-. 0.15 --
(.+-.)-Z-BDDA.sup..paragraph. 318.2 .+-. 21.8 -0.023 .+-.
0.027.sup.1 30.1 .+-. 2.8.sup.1 1.3 .+-. 0.1 2.04 .+-. 0.45.sup.1
-- (+)-Z-BDDA.sup..paragraph. 315.2 .+-. 18.4 0.045 .+-.
0.017.sup.4 35.1 .+-. 8.6.sup.1 2.1 .+-. 0.3.sup.1,3 2.57 .+-.
0.79.sup.1,2 -- Male control 402.8 .+-. 25.0 0.134 .+-. 0.013 82.1
.+-. 6.1 5.4 .+-. 0.7 3.82 .+-. 0.19 0.64 .+-. 0.10
vehicle.sup..dagger. 339.8 .+-. 22.6 0.087 .+-. 0.011 81.5 .+-. 3.9
8.2 .+-. 0.7.sup.1 3.55 .+-. 0.11 0.56 .+-. 0.03
estradiol.sup..paragraph. 298.2 .+-. 18.8.sup.1 -0.057 .+-.
0.012.sup.1,2 51.5 .+-. 9.5.sup.1,2 4.2 .+-. 0.4.sup.2 0.94 .+-.
0.13.sup.1,2 0.09 .+-. 0.01.sup.1,2 (.+-.)-Z-BDDA.sup..paragr- aph.
344.6 .+-. 15.5 -0.099 .+-. 0.009.sup.1,2 36.2 .+-. 2.6.sup.1,2 4.5
.+-. 0.4.sup.2 0.72 .+-. 0.06.sup.1,2 0.08 .+-. 0.02.sup.1,2
(+)-Z-BDDA.sup..paragraph. 326.0 .+-. 9.5.sup.1 -0.174 .+-.
0.027.sup.1,2,3,4 24.6 .+-. 2.2.sup.1,2,3 5.1 .+-. 0.2.sup.2 0.84
.+-. 0.06.sup.1,2 0.13 .+-. 0.04.sup.1,2,3 *All values are the mean
.+-. SEM, n = 5 animals/treatment (9-10 weeks of age).
.sup..dagger.10% ethanol-90% olive oil solution.
.sup..paragraph.Treatment groups received the compound (2.5 ug/g of
body weight) in 10% ethanol-90% olive oil solution.
.sup.1significantly different from control (p < 0.05);
.sup.2significantly different from vehicle (p < 0.05);
.sup.3significantly different from estradiol (p < 0.05);
.sup.4significantly different from (.+-.)-Z-BDDA (p < 0.05).
[0232]
2TABLE 2 Experiment 2. The effects of (-)-, (+)-, and
(.+-.)-Z-bisdehydrodoisynolic acids (Z-BDDA) and
(+)-17.beta.-estradiol on metabolic and reproductive parameters in
male and female rats on treatment for 5-6 weeks* Blood glucose
Uterus/testis Prostate Treatment Food intake (g) FER (food
efficiency ratio) (mg/dl) weight (g) weight (g) Female
vehicle.sup..dagger. 625.8 .+-. 49.3 0.071 .+-. 0.007 107.00 .+-.
11.47 1.22 .+-. 0.21 -- estradiol.sup..paragraph. 699.6 .+-. 33.7
0.039 .+-. 0.004 100.80 .+-. 10.22 4.83 .+-. 1.30.sup.1 --
(.+-.)-Z-BDDA.sup..paragraph. 704.0 .+-. 51.7 0.017 .+-.
0.003.sup.1,2 80.40 .+-. 5.66 4.25 .+-. 1.04.sup.1 --
(+)-Z-BDDA.sup..paragraph. 693.6 .+-. 72.3 0.026 .+-. 0.008.sup.1
89.60 .+-. 6.15 4.02 .+-. 0.91.sup.1 -- (-)-Z-BDDA.sup..paragraph.
675.2 .+-. 37.9 0.011 .+-. 0.005.sup.1,2 95.00 .+-. 8.33 1.44 .+-.
0.34.sup.2,3,4 -- Male vehicle.sup..dagger. 525.8 .+-. 27.3 0.12
.+-. 0.02 134.80 .+-. 10.97 2.68 .+-. 0.26 0.25 .+-. 0.11
estradiol.sup..paragraph. 590.4 .+-. 47.7 0.00 .+-. 0.01.sup.1
108.00 .+-. 10.90 0.74 .+-. 0.04.sup.1 0.06 .+-. 0.02
(.+-.)-Z-BDDA.sup..paragraph. 530.0 .+-. 33.0 -0.04 .+-.
0.01.sup.1,2 98.60 .+-. 14.00 0.78 .+-. 0.02.sup.1 0.16 .+-. 0.02
(+)-Z-BDDA.sup..paragraph. 676.0 .+-. 33.2 -0.02 .+-. 0.01.sup.1
94.00 .+-. 10.18.sup.1 0.61 .+-. 0.02.sup.1 0.02 .+-. 0.00.sup.1
(-)-Z-BDDA.sup..paragraph. 650.0 .+-. 54.3 -0.01 .+-. 0.01.sup.1
88.00 .+-. 2.00.sup.1 0.57 .+-. 0.04.sup.1 0.03 .+-. 0.10.sup.1
*All values are the mean .+-. SEM, n = 5 animals/treatment (9-10
weeks of age). .sup..dagger.10% ethanol-90% olive oil solution.
.sup..paragraph.Treatment groups received the compound (2.5 ug/g of
body weight) in 10% ethanol-90% olive oil solution.
.sup.1significantly different from vehicle (p < 0.05);
.sup.2significantly different from estradiol (p < 0.05);
.sup.3significantly different from (.+-.)-Z-BDDA(p < 0.05);
.sup.4significantly different from (+)-Z-BDDA (p < 0.05).
[0233] Parallel to their effects on reproductive parameters, E2 and
the three forms of Z-BDDA also elicited similar and distinct
effects on metabolic parameters in the male and female rats. For
example, in the female rats in both experiments, (-)-and
(.+-.)-Z-BDDA appeared to repress weight gain slightly more than
did (+)-Z-BDDA and E2, while in the male rats estradiol and the
three Z-BDDA forms caused a dramatic reduction in body weight
(FIGS. 2 and 3).
[0234] Surprisingly, the specific enantiomers of Z-BDDA appeared to
elicit a divergence between estrogenicity and weight repression in
the female rats (FIGS. 1, 2, and 3 ), whereas in the male rats, all
three Z-BDDA forms not only elicited estrogenic-like effects (Table
1 and 2), but appeared to be more potent with respect to weight
repression (FIGS. 2 and 3). The male rats exhibited a reduction in
visceral fat weight when treated with E2 and each of the three
Z-BDDA forms, respectively, versus vehicle (Table 1). It is
apparent from other metabolic parameters (i.e., food intake and
FER) that the weight repression in weight-gain and even the weight
reduction were independent of food intake (Table 1 and 2). In fact,
almost all of the difference in weight can be accounted for by a
decrease in food efficiency (Table 1 and 2).
[0235] In addition to the gross physiological parameters, metabolic
processes were also examined. The female rats receiving (+)-Z-BDDA,
(.+-.)-Z-BDDA, or E2 exhibited a decrease in total cholesterol
versus control (Table 1). The males exhibited a more pronounced
cholesterol-lowering pattern (p<0.05), (.+-.)-Z-BDDA having the
most profound effect (Table 1). Furthermore, in the second
experiment, male rats receiving either (-)-or (+)-Z-BDDA compounds
exhibited a reduction (p<0.05) in blood glucose, while the
(.+-.)-Z-BDDA treated males and the females treated with all the
Z-BDDA compounds demonstrated a trend (p=0.06) toward a reduction
in blood glucose (Table 2).
[0236] In the same experiment, luteinizing hormone, prolactin, and
testosterone were measured to detect possible endocrine disruption
caused by the Z-BDDA compounds. No significant changes in
luteinizing hormone and prolactin were observed. However, compared
to the vehicle, (.+-.)-Z-BDDA as well as E2 caused a significant
(p<0.05) testosterone suppression in the male rats, a trend
which was less pronounced with (-)- and (.+-.)-Z-BDDA.
Conclusions
[0237] These results with the Z-BDDA compounds demonstrate that
specific enantiomers of Z-BDDA appear to confer cardioprotective
benefits (i.e., reduction in cholesterol, body weight, blood
glucose, and positive alterations in distribution of visceral fat).
Wilson P W: "The Impact of Estrogen on Cardiovascular Disease."
Perspective Studies: The Framingham Study. Postgrad Med 51-53:89-90
(1989); Cooper R L, Kavlock R J: "Endocrine Disruptors and
Reproductive Development: A Weight-of-Evidence Overview." J
Endocrinol 152:159-166 (1997); and Reubinoff B E, Wurtman J,
Rojansky N. Adler D, Stein P, Schenker J G, BrZeZinski A: "Effects
of Hormone Replacement Therapy on Weight, Body Composition, Fat
Distribution, and Food Intake in Early Postmenopausal Women: A
Prospective Study." Fertil Steril 64:963-968 (1995). The (-)
enantiomer appears to minimize undesirable estrogenic effects on
reproductive tissues. The Z-BDDA compounds exhibited a
cholesterol-lowering effect consistent with that elicited by other
estrogenic compounds. See Heer J, Billeter J R, Miescher K:
"Totalsynthese der racemischen bisdehydro-doisynolsure. ber
oestrogene carbosuren IV." Helv. Chim. Acta 28:1342-1354 (1945); Ke
H Z, Chen H A, Simmons H A, Qi H, Crawford D T, Pirie C M,
Chidsey-Frink K L, Ma Y F, Jee W S S, Thompson D D: "Comparative
Effects of Droloxifene, Tamoxifen, and Estrogen on Bone, Serum
Cholesterol, and Uterine Histology in the Ovariectomized Rat
Model." Bone 20:31-39 (1997); Sato M, Rippy M K, Bryant H U:
"Raloxifene, Tamoxifen, Nafoxidine, or Estrogen Effects on
Reproductive and Nonreproductive Tissues in Ovariectomized Rats."
FASEB J 10:905-912 (1996); Dodge J A, Glasebrook A L, Magee D A,
Phillips D L, Sato M, Short LL, Bryant H U: "Environmental
Estrogens: Effects on Cholesterol Lowering and Bone in the
Ovariectomized Rat." J Steroid Biochem Molec Biol 59:155-161(1996);
and Hart J E: "Endocrine Pathology of Estrogens: Species
Differences." Pharmac Ther 47:203-218 (1990). Surprisingly, the
hypocholesterolemic, weight-repressing, and visceral fat-reducing
effects were demonstrated in reproductively intact male and female
rats. This effect may be unique among non-steroidal estrogens. For
example, raloxifene has no significant clinical effects in healthy,
menstruating women. Heywood R, Wadsworth P F: "The Experimental
Toxicology of Estrogens." Pharmac Ther 8:125-142 (1980). While
raloxifene as well as tamoxifen and nafoxidine seem to elicit a
cholesterol-lowering and a minimal weight-repressing effect in
ovariectomized animals, the Z-BDDA compounds appear to be much more
effective and, moreover, produce this effect in reproductively
intact animals. Meyers C Y, Lutfi H G, Adler S: "Transcriptional
Regulation of Estrogen-Responsive Genes by Non-Steroidal Estrogens:
Doisynolic and Allenolic acids." J Steroid Biochem Molec Biol
62:477-489 (1997); Heer J, Billeter J R, Miescher K: "Totalsynthese
der racemischen bisdehydro-doisynolsure. ber oestrogene carbosuren
IV." Helv. Chim. Acta 28:1342-1354 (1945); Ke H Z, Chen H A,
Simmons H A, Qi H, Crawford D T, Pirie C M, Chidsey-Frink K L, Ma Y
F, Jee W S S, Thompson D D: "Comparative Effects of Droloxifene,
Tamoxifen, and Estrogen on Bone, Serum Cholesterol, and Uterine
Histology in the Ovariectomized Rat Model." Bone 20:31-39 (1997);
Sato M, Rippy M K, Bryant H U: "Raloxifene, Tamoxifen, Nafoxidine,
or Estrogen Effects on Reproductive and Nonreproductive Tissues in
Ovariectomized Rats." FASEB J 10:905-912 (1996); and Heywood R,
Wadsworth P F: "The Experimental Toxicology of Estrogens." Pharmac
Ther 8:125-142 (1980). Being observed in intact, non-castrate male
and female animals, these effects suggest clinical applications for
these or similar compounds in treating pre- as well as
post-menopausal women, and males at risk for cardiovascular and
prostatic disease.
[0238] The distinct effects elicited by all three Z-BDDA forms on
body weight, food intake, FER, and visceral fat appear to be
compound-specific and somewhat divergent from the effects elicited
by E2. It is apparent from the food intake and FER data that the
repression in body-weight gain was independent of the quantity of
food consumed. The fact that almost all of the variation in weight
can be accounted for by a decrease in food efficiency points to a
metabolic alteration elicited by the Z-BDDA compounds rather than
appetite suppression as the weight-repressing mechanism. This
finding is in contrast to the effects of other estrogenic compounds
on body weight. While some other estrogens may cause weight
repression, in those cases it appears to be compound-, species-,
and gender-specific and, in sharp contrast to the results obtained
with the present BDDA compounds, can be explained by a reduction in
food intake. In further contrast to the present results with the
BDDA compounds, these effects elicited by other estrogens are
reported in studies with castrated rather than reproductively
intact animals. See Heer J, Billeter J R, Miescher K:
"Totalsynthese der racemischen bisdehydro-doisynolsure. ber
oestrogene carbosuren IV." Helv. Chim. Acta 28:1342-1354 (1945); Ke
H Z, Chen H A, Simmons H A, Qi H, Crawford D T, Pirie C M,
Chidsey-Frink K L, Ma Y F, Jee W S S, Thompson D D: "Comparative
Effects of Droloxifene, Tamoxifen, and Estrogen on Bone, Serum
Cholesterol, and Uterine Histology in the Ovariectomized Rat
Model." Bone 20:31-39 (1997); Sato M, Rippy M K, Bryant H U:
"Raloxifene, Tamoxifen, Nafoxidine, or Estrogen Effects on
Reproductive and Nonreproductive Tissues in Ovariectomized Rats."
FASEB J 10:905-912 (1996); Dodge J A, Glasebrook A L, Magee D A,
Phillips D L, Sato M, Short L L, Bryant H U: "Environmental
Estrogens: Effects on Cholesterol Lowering and Bone in the
Ovariectomized Rat." J Steroid Biochem Molec Biol 59:155-161
(1996); and Hart J E: "Endocrine Pathology of Estrogens: Species
Differences." Pharmac Ther 47:203-218 (1990).
[0239] These results also suggest that (-)-Z-BDDA appears to
exhibit both estrogenic and anti-estrogenic activities in female
rats. This was not the case for the males, and may be dependent on
the interaction of (-)-Z-BDDA with endogenous E2. Racemic Z-BDDA
and its two enantiomers, while all promoting weight-repressing
effects in female rats, differed in their capacity to elicit
uterotropism, a classic assay for estrogenic activity.
Surprisingly, (-)-Z-BDDA did not induce the significant increases
in uterine weight observed with (+)- or (.+-.)-Z-BDDA or E2. In
contrast to the results observed in the chronic treatment study, it
has previously been demonstrated that the (-) enantiomer of
(.+-.)-Z-BDDA is the enantiomer responsible for the high
uterotropic activity observed when administered acutely. Anner G,
Miescher K: Hydrierungs-Und Umlagerungs-Reaktion in der
Doisynolsure-Reihe. Oestrogene Carbonsuren XII. Helv. Chim. Acta 29
(1946) 1889-1895; Die totalsyntheses von racemischen doisynolsuren
XXI. ber oestrogene carbonsueren. ibid 30:1422-1432 (1947); Tschopp
E: "Wirksamkeit, organconzentration und ausscheidung der
7-methyl-bisdehydro-doisynolsure." Helv Physiol Pharmacol Acta
4:401-410 (1946); Tschopp E: "Die oestrogene wirkung der
bisdehydrodoisynolsure und ihre derivate." Helv Physiol Pharmacol
Acta 4:271-284 (1946); Rometsch R, Miescher K: "Die spaltung des
racemates der n-bisdehydro-doisynolsure. ber ostrogene
carbonsaiuren X." Helv Chim Acta 29:1231-1235 (1946); and Terenius
L: "Differential Inhibition In Vitro of 17.beta.-Estradiol Binding
in the Mouse Uterus and Vagina by Optical Antipodes of Estrogen."
Molec Pharmac 4:301-310 (1968). The basis of the difference between
the results presented herein and those reported previously is not
known. However, in addition to differences in duration of
treatment, other factors that may have contributed to the lack of
uterotropism elicited by (-)-Z-BDDA in the present studies include
the dosages used and the species and ages of the animals.
[0240] Of further interest are the differences observed in the
potency of the Z-BDDA compounds when the in vivo results are
compared with either cell-culture assays measuring activation of
estrogen receptor, or with in vitro assays of relative
receptor-binding affinity. Numerous competitive binding-inhibition
studies with the classical estrogen receptors (ER.alpha.) have
demonstrated that the binding affinity of (.+-.)-Z-BDDA is much
lower than that of estradiol. Meyers C Y, Kolb V M, Gass G H, Rao B
R, Roos C F, Dandliker W B: "Doisynolic-Type Acids--Uterotropically
Potent Estrogens which Compete Poorly with Estradiol for Cytosolic
Estradiol Receptors. J Steroid Biochem 31:393-404 (1988); Soto A M,
Meyers C Y, Sonnenschein C: "How Many Rings Can be Cleaved from a
Steroidal Estrogen While Preserving its Estrogenic Activity?"The
Endocrine Society, 70th Annual Meeting, Abstract (1988); and Banz
J, Winters T A, Hou Y, Adler S, Meyers C Y: "Activities of
Non--Classical Estrogens: Effects of (-)-, (.+-.)-, and
(.+-.)-Z-Bisdehydrodoisynolic Acids In Vitro and on Body Weight in
Male and Female Rats." The Endocrine Society, 80th Annual Meeting,
Abstract (1998). These results were substantiated recently by
direct binding studies using preparations of human ER.alpha., and
are in agreement with previous results with (-)-Z-BDDA, which were
determined with mouse uterine tissue in competitive
binding-inhibition studies. Terenius L: "Differential Inhibition In
Vitro of 17.beta.-Estradiol Binding in the Mouse Uterus and Vagina
by Optical Antipodes of Estrogen." Molec Pharmac 4:301-310 (1968);
and Meyers C Y, Lutfi HG, Adler S: "Transcriptional Regulation of
Estrogen-Responsive Genes by Non-Steroidal Estrogens: Doisynolic
and Allenolic acids." J Steroid Biochem Molec Biol 62:477-489
(1997). Hence, there is an apparent activity/binding paradox,
suggesting that the classic estrogen receptor, ER.alpha., may not
be the exclusive receptor or pathway mediating the actions of
Z-BDDA compounds, or possibly even those of estradiol. Meyers C Y,
Kolb V M, Gass G H, Rao B R, Roos C F, Dandliker W B:
"Doisynolic-Type Acids--Uterotropically Potent Estrogens which
Compete Poorly with Estradiol for Cytosolic Estradiol Receptors. J
Steroid Biochem 31:393-404 (1988).
[0241] Recently, a new form of estrogen receptor, ERA, has been
identified, and its role in estrogenic regulation in various target
tissues and its affinity for non-steroidal ligands are currently
being defined. Kuiper G G, Carlsson B, Grandien K, Enmark E,
Haggblad J, Nilsson S, Gustafsson J: "Comparison of the Ligand
Binding Specificity and Transcript Tissue Distribution of Estrogen
Receptors .alpha. and .beta.." Endocrinology 138:863-870 (1997);
and Pace P, Taylor J, Suntharalingam S, Coombes R C, Ali S: "Human
Estrogen Receptor .beta. Binds DNA in a Manner Similar to and
Dimerizes with Estrogen Receptor .alpha.." J Biol Chem
272:25832-25838 (1997). Initial studies comparing the classical
ER.alpha. and the novel estrogen receptor ER.beta. show very
similar results. The binding affinity of (+)-Z-BDDA is even lower
than that of the (-) enantiomer, and both enantiomers have a much
lower affinity for estrogen receptors than does estradiol, whether
measured via direct receptor binding assays or by generating
dose-response profiles using activation of estrogen-responsive
reporter genes in cell-culture systems. Banz J, Winters TA, Hou Y,
Adler S, Meyers C Y: "Activities of Non--Classical Estrogens:
Effects of (-)-, (+)-, and (.+-.)-Z-Bisdehydrodoisynolic Acids In
Vitro and on Body Weight in Male and Female Rats." The Endocrine
Society, 80th Annual Meeting, Abstract (1998). The evaluation of
ER.beta. has not resolved the apparent paradox. However, the use of
heterodimers of ER.alpha. and ER.beta. has not been evaluated, and
may add a further degree of complexity to this binding/activity
evaluation.
[0242] Alternatively, there is evidence that in vivo, serum-binding
proteins could account for part of the activity/binding paradox
emanating from a comparison of estradiol on one hand and the three
Z-BDDA forms on the other. DanZo B J: "Environmental Xenobiotics
May Disrupt Normal Endocrine Function by Interfering with the
Binding of Physiological Ligands to Steroid Receptors and Binding
Proteins." Environ Health Perspect 105:294-301 (1997); and Nagel S
C, vom Saal F S, Thayer K A, Dhar M G, Boechler M, Welshons W V:
"Relative Binding Affinity--Serum Modified Access Assay Predicts
the Relative In Vivo Bioactivity of the Xenoestrogens Bisphenol A
and Octylphenol." Environ Health Perspect 105:70-76 (1997).
Steroid-hormone binding globulin (SHBG) and serum albumin appear to
have a much higher affinity for estradiol than for many
environmental and synthetic estrogens. Thus, in vivo, there is a
relatively higher level of free versus bound compound compared to
estradiol than would be predicted from in vitro binding studies
alone. In addition, nonsteroidal environ-mental and synthetic
estrogens may also elicit biological effects independent of the
ligand-estrogen receptor complex (i.e., antioxidant and enzyme
modulation). Wehling M: "Specific, Nongenomic Actions of Steroid
Hormones." Annu Rev Physiol 59:365-393 (1997); Akiyama T, Ishida J,
Nakagawa S, Ogawara H, Watanabe S, Itoh N, Shibuya M, Fukami Y:
"Genistein, A Specific Inhibitor of Tyrosine--Specific Protein
Kinases." J Biol Chem 262:5592-5595 (1987); Peterson G, Barnes S:
"Genistein Inhibits Both Estrogen and Growth Factor--Stimulated
Proliferation of Human Breast Cancer Cells. Cell Growth &
Differentiation 7:1345-1351 (1996); Spink D C, Johnson J A, Connor
S P, Aldous K M, Gierthy J F: "Stimulation of 17 Beta-Estradiol
Metabolism in MC F-7 Cells by Bromochloro-and
Chloromethyl--Substituted DibenZo-p-dioxins and Dibenzofurans:
Correlations with Antiestrogenic Activity." Journal of Toxicology
& Environmental Health 41:451-466 (1994); Behl C, Skutella T,
LeZoualch F, Post A, Widmann M, Newton C J, Holsboer F:
"Neuroprotection Against Oxidative Stress by Estrogens:
Structure-Activity Relationship." Mol Pharmacol 51:535-541 (1997);
Wiseman H, O'Reilly J: "Oestrogens as Antioxidant
Cardioprotectants." Biochemical Society Transactions 25:54-59
(1997); and Smith C L, Conneely O M, O'Malley B W: "Modulation of
the Ligand-Independent Activation of the Human Estrogen Receptor by
Hormone and Antihormone." Proc Natl Acad Sci 90:6120-6124 (1993).
The final in vivo effect of these compounds may reflect all of
these contributions.
[0243] The foregoing data, generated in intact, non-castrated male
and female animals, indicate that the observed effects, unlike
those reported in comparable studies with tamoxifen, nafoxidine, or
raloxifene, are not obscured by endogenous estradiol. Heer J,
Billeter J R, Miescher K: "Totalsynthese der racemischen
bisdehydro-doisynolsure. ber oestrogene carbosuren IV." Helv. Chim.
Acta 28:1342-1354 (1945); Ke H Z, Chen H A, Simmons H A, Qi H,
Crawford D T, Pirie C M, Chidsey-Frink K L, Ma Y F, Jee W S S,
Thompson D D: "Comparative Effects of Droloxifene, Tamoxifen, and
Estrogen on Bone, Serum Cholesterol, and Uterine Histology in the
Ovariectomized Rat Model." Bone 20:31-39 (1997); Sato M, Rippy M K,
Bryant H U: "Raloxifene, Tamoxifen, Nafoxidine, or Estrogen Effects
on Reproductive and Nonreproductive Tissues in Ovariectomized
Rats." FASEB J 10:905-912 (1996); and Heywood R, Wadsworth P F:
"The Experimental Toxicology of Estrogens." Pharmac Ther 8:125-142
(1980). The Z-BDDA compounds cause weight repression/reduction in
male and female rats via an unknown mechanism. The results
demonstrate remarkable selective estrogen receptor modulator (SERM)
activity, and strongly suggest clinical applications for these
compounds in peri- as well as post-menopausal women. Furthermore,
they suggest clinical applications for these compounds (or
appropriate derivatives thereof) in males at risk for
cardiovascular and prostatic disease.
Example 3
Effects of Z-Bisdehydrodoisynolic Acids on In situ Apoptosis in
Primary Porcine Granulosa Cells
[0244] Estrogens have been found to decrease ovarian follicle
atresia (Tilly et al. (1991) Endocrinology 129:2799-2801), which in
turn could increase the number of follicles recruited and thus
ovulated each menstrual or estrous cycle in humans and animals,
respectively. Apoptosis, or programmed cell death, is the
underlying mechanism for follicular atresia. This experiment was
performed to determine if BDDAs affect follicular cell
apoptosis.
[0245] Materials and Methods
[0246] Tissue Culture
[0247] Porcine ovaries were obtained from local packing plants and
transported to the laboratory on ice-cold Hank's balanced salt
solution (HBSS). Each of the follicles was aspirated with an
insulin syringe, and the follicular fluid was centrifuged at 3000
rpm and 4.degree. C. for 15 min. The supernatant was poured off and
the cells were washed in 5 mls of cold HBSS and centrifuged for
another 10 min. The cells were again resuspended in HBSS, and the
number of viable cells counted under the microscope using a
hematocytometer. Once the number of cells was determined, the cells
were centrifuged again for 10 min. and resuspended in the
appropriate volume of Eagle's minimum essential media (MEM)
containing 10% fetal bovine serum (FBS) and
antibiotic/antimycotics. The cells were plated at 250,000 per well
in 8 chamber microscope slides (Nunc, Naperville, Ill.) which were
pre-treated with poly-L-lysine for 10 min. The slides were
incubated at 5% CO.sub.2/95% air at 37.degree. C.. Approximately 12
hours later, the medium was removed by vacuum and replaced with
pre-warmed, serum-free MEM. Cells were then treated for 2-3 hrs
with MEM only to wash out any estrogen effects from the serum
(Winters et al. (1994) Biol. Reprod. 50 (Suppl. 1):113; Suttner et
al. (1998) Biol. Reprod. 59 (Suppl.): (Accepted for
publication).
[0248] Treatments
[0249] Following serum-free MEM treatment, cells were treated for
24 hr with (+) or (-) enantiomers of Z-BDDA or estradiol at 0.1, 1,
and 10 .mu.M, or EtOH vehicle control in serum-free MEM.
[0250] Apoptosis Assay
[0251] Cells were subsequently processed using an in situ apoptosis
assay kit (Apotag-Plus In Situ Apoptosis Kit--Peroxidase, Edition
1.1., Oncor, Gaithersburg, Md., 1995) to study ovarian apoptosis
(Suttner et al., 1998). The slides were washed in two changes of
PBS for 5 min each, and then quenched in 2% hydrogen peroxide in
methanol for 5 min. at room temperature. After pre-treatment with
an equilibration buffer, 13 ul of Terminal deoxynucleotidyl
Transferase (TdT) diluted with reaction buffer and distilled water
were added, and the slides were incubated for 1 h at 37.degree. C.
in a humidified chamber. After this 1 h period, the slides were put
in pre-warmed stop wash buffer in the incubator for 30 min to stop
the reaction. Next, the slides were washed in three changes of PBS
for 5 min. each, and the anti-digoxigenin peroxidase was placed on
the slides for 30 min. in a humidified chamber at room temperature.
Once this time was up, the slides were washed in four changes of
PBS for 5 min. each and stained with diaminobenzidine substrate
solution for 15-20 min. This yielded a brown stain in apoptotic
cells. After washing in three changes of distilled water for 1
min., followed by a 5 min. wash, the slides were counterstained in
methyl green for 8 min. This yielded a blue/green stain in
non-apoptotic cells. Once counterstained, the specimens were washed
in three changes of distilled water and 100% butanol, respectively,
by dipping 10 times in the first and second washes, followed by 30
sec. in the third wash. The slides were cleared in three washes of
xylene for 2 min. each and then mounted under coverslips with
permount.
[0252] Image Analysis
[0253] The degree of apoptosis for the colorimetric apoptosis assay
was quantified microscopically using an image analysis system
(Optimas 5.23, Optimas Users Guide, 5th Edition, Redmond, Wash.).
Ten measurements (captured images) were taken for each
concentration based on a pre-determined grid. Brown and blue/green
color thresholds for apoptotic and non-apoptotic cells,
respectively, were set for each captured image. Percentage area of
each color was then quantitated using the image analysis system.
Data were then transferred to a spreadsheet (Excel, Microsoft
Corp., Redmond, Wash.) for sorting before statistical analysis.
This procedure was repeated for each slide and each concentration
in duplicate.
[0254] Statistics
[0255] Statistical analysis was performed using a statistical
program (SAS, 1988, SAS/STAT User's Guide. Statistical Analysis
Institute, Cary, N.C.). Contrast analyses were run for the weekly
experiments, and all of the treatments were compared. The level of
significance was determined at p<0.05.
Results
[0256] (-)-Z-BDDA treatment decreased (P<0.01) mean apoptosis (%
are.sup.a) from 63.4% in the controls to 26.1% in treated cells.
(+)-Z-BDDA treatment did not appreciably change mean apoptosis in
the controls (68.4%) vs. the treated cells (61.1%). Estradiol
treatment combined was not different from controls; however,
percent apoptosis was lower (P<0.05) at 10 .mu.M estradiol
(23.8%). In addition, (-)-Z-BDDA tended to decrease (P=0.06)
percent apoptosis vs. estradiol (46.5).
Conclusions
[0257] These results indicate that (-)-Z-BDDA has the ability to
decrease apoptosis in granulosa cells from the ovarian follicle of
a porcine experimental model. Decreased follicle apoptosis could
lead to more follicle recruitment and ovulations in the mammalian
ovary. The inhibition of granulosa cell apoptosis by (-)-Z-BDDA
appears to be more substantial than that of estradiol. The
(+)-enantiomer did not appear to have an effect in these
experiments. However, (+)-Z-BDDA could be active at a higher
concentration, or be acting as an antiestrogen inhibiting the
estrogenic effect seen with (-)-Z-BDDA and estradiol. These results
suggest that the use of (-)-Z-BDDA in human and/or veterinary
medicine could lead to more follicle recruitment and ovulations,
thus increasing fertility. (+)Z-BDDA may may have applications as a
birth control drug. In addition, the BDDAs could potentially be
used to modulate other physiological processes controlled by
apoptosis, including maturation of the immune system, embryonic
development, luteolysis, male pattern baldness, cancer, tissues
responding to thermal and metabolic stress, tissues responding to
hormonal stimuli (especially estrogens), and normal tissue turnover
(Bowen et al. (1990) Programmed Cell Death in Tumors and Tissues,
Chapman & Hall, New York, N.Y.).
Example 4
Differential Effects of Estrogenic Carboxylic Acids on the Prostate
and Testis of Male Rats
[0258] Estrogens have been used in the treatment of prostate
cancer; however, these estrogens have negative feminizing side
effects. These include shrinkage of the testis and accessory glands
(including the prostate), gynecomastia, salt and water retention,
and inhibition of other secondary male sex characteristics
(including loss of libido and impotence). Gudziak, M R, and A Y
Smith. "Hormonal Therapy for Stage D Cancer of the Prostate" West J
Med 160:351-359 (1994). In addition, estrogen therapy in males
leads to a three-fold increased risk of thromboembolic events
(including heart attacks, strokes, and blood clots). Glashan, R W,
and M R G Robinson. "Cardiovascular Complications in the Treatment
of Prostatic Carcinoma." Br J Urol 53:624-627 (1981). Since
estrogen treatment in males causes these undesirable effects,
estrogens are only used in severe prostate carcinoma, and are not
usually used in other prostatic conditions such as benign prostate
hypertrophy. Jacobi, G H. "Hormonal Treatment of Metastatic
Carcinoma." In: The Prostate, pp. 119-128.(J M Fitzpatrick and R J
Krane, eds., Churchill Livingstone, New York, N.Y. 1989); de Klerk,
D P, and F Allen. "Medical Therapy for Benign Prostatic
Hyperplasia." In: The Prostate, pp. 119-128 (J M FitZpatrick and R
J Krane, eds., Churchill Livingstone, New York, N.Y. 1989).
[0259] This study was undertaken to determine the effects that
enantiomers of the estrogenic carboxylic acids,
Z-bisdehydrodoisynolic acids (BDDA) and hydroxyallenolic acids
(HAA), have on the prostate, testis, and other physiological
parameters in male rats. As reported below, the results demonstrate
that these compounds possess utility as a therapy for prostatic
disease, as well as in other clinical applications in males.
[0260] Materials and Methods Sixty male Sprague-Dawley rats, 7-8
weeks of age, were randomly assigned to groups of ten animals. Each
group was randomly assigned to one of six treatments: Vehicle
control (C), Estradiol-17.beta. (E), (-)-Z-BDDA, (+)-Z-BDDA,
(-)-HAA, and (+)-HAA. The compounds were all administered at a dose
of 0.1 .mu.g/g of body weight in 0.1 cc once a day for 6 weeks. The
estrogenic compounds were dissolved in 10% ethanol and 90% olive
oil vehicle. A temperature of 21.degree. C. and an artificial 12 h
light-dark cycle were maintained in the animal room. All animals
were maintained on standard chow in powdered form for six weeks,
and then sacrificed after an overnight fast under i.p.
pentobarbitol anesthesia (50 mg/kg). Animal weight was measured
weekly during the study. During sacrifice, blood was collected via
cardiac puncture. Immediately following sacrifice, the fat pads,
livers, pituitaries, testes, seminal vesicles, and prostate were
removed and weighed, and snap frozen in liquid nitrogen. Prostates
and one testis from 2-3 animals in each treatment group were fixed
in 10% formalin for histological examination. These tissues were
fixed overnight, blocked in paraffin, sectioned at 4 .mu.m, stained
with Hematoxylin and Eosin, cover slipped, and examined
microscopically. Quantitative results were subjected to an analysis
of variance and means separated by a Tukey's Test (SYSTAT, Chicago,
Ill.).
[0261] Results
[0262] Rats in all five estrogen treatments showed a significant
decrease (P<0.05) in weight gain compared to that in rats in the
control (C) group (Table 3). The (+)-Z-BDDA-treated rats gained
more (P<0.05) weight than the estradiol-treated,
(-)-Z-BDDA-treated, (-)-HAA-treated, and (+)-HAA-treated rats. The
(-)-Z-BDDA-treated rats had the lowest weight change, and was lower
(P<0.05) than that in the control, estradiol-treated,
(+)-Z-BDDA-treated, and (+)-HAA-treated groups.
[0263] Prostate weights as a percentage of bodyweight were lower
(P<0.05) than that in controls in all five estrogen treatments
(Table 3). The weights of testes and seminal vesicles as a
percentage of bodyweight were lower (P<0.05) than that of
control rats in the estradiol-treated, (-)-Z-BDDA-treated,
(-)-HAA-treated, and (+)-HAA-treated rats (Table 1). The
(+)-Z-BDDA-treated rats did not have significantly smaller testes
or seminal vesicles as a percentage of bodyweight, although gross
testes weights unadjusted for bodyweight were lighter (P<0.05)
than those in control rats (data not shown). There were no obvious
signs of gynecomastia in any of the rats.
3TABLE 3 The effects of (-)- and (+)-Z-bisdehydrodoisynolic acids
(BDDA), (-)- and (+)-hydroxyallenolic acid (HAA), and
(+)-17.beta.-estradiol (E) on metabolic and reproductive parameters
in male rats on treatment for 6 weeks* Testis Weight as % Prostate
Weight as % Seminal Vesicle Weight as % Treatment Body Weight (g)
Body Weight Body Weight Body Weight vehicle.sup..dagger. 336.3 .+-.
4.9 1.14 .+-. 0.05 0.16 .+-. 0.02 0.21 .+-. 0.03 E.sup..paragraph.
207.2 .+-. 4.6.sup.1 0.49 .+-. 0.13.sup.1,4 0.05 .+-. 0.00.sup.1
0.02 .+-. 0.00.sup.1 (-)-BDDA.sup..paragraph. 166.5 .+-.
4.5.sup.1,2,4 0.31 .+-. 0.02.sup.1,4 0.08 .+-. 0.00.sup.1 0.06 .+-.
0.02.sup.4 (+)-BDDA.sup..paragraph. 234.8 .+-. 10.1.sup.1,2 0.98
.+-. 0.15 0.05 .+-. 0.01.sup.1 0.23 .+-. 0.12.sup.2
(-)-HAA.sup..paragraph. 180.9 .+-. 4.5.sup.1,2,4 0.31 .+-.
0.01.sup.1,4 0.08 .+-. 0.01.sup.1 0.04 .+-. 0.00.sup.1,4
(+)-HAA.sup..paragraph. 196.2 .+-. 5.2.sup.1,4,5 0.38 .+-.
0.02.sup.1,4 0.08 .+-. 0.01.sup.1 0.10 .+-. 0.04 *A11 values are
the mean .+-. SEM, n = 10 animals/treatment (.about.7 weeks of
age). .sup..dagger.0% ethanol-90% olive oil solution.
.sup..paragraph.Treatment groups received the compound (0.1 .mu.g/g
of body weight) in 10% ethanol-90% olive oil solution.
.sup.1significantly different from vehicle (p < 0.05);
.sup.2significantly different from estradiol (p < 0.05);
.sup.3significantly different from (-)-BDDA (p < 0.05);
.sup.4significantly different from (+)-BDDA (p < 0.05);
.sup.5significantly different from (-)-HAA (p < 0.05).
[0264] Histological examination of the prostate showed normal
alveoli in the control and (+)-Z-BDDA-treated rats, with the
tubules and alveoli being slightly smaller only in the
(+)-Z-BDDA-treated rats (FIGS. 4a-f). However, the alveoli showed
significant degrees of atrophy in the other four treatments, with
the (-)-HAA-treated rats displaying the largest degree of atrophy.
In the testis, spernatogenesis and Leydig cells were normal in the
control and (+)-Z-BDDA-treated rats, but were severely attenuated
in rats in the other four groups (FIGS. 5a-f). Rats in these four
treatment groups had spermatogenesis halted in late meiosis, early
spermiogenesis. The estradiol-treated rats showed spermatogenesis
halted at round (Golgi phase) spermatids, and Leydig cells were
small. The (-)-Z-BDDA-treated rats were halted primarily at the
secondary spermatocyte phase, with a few spermatogenic cells
reaching round spermatid. The (-)-Z-BDDA-treated rats also had
severely atrophied Leydig cells, the smallest of all the
treatments. The (-)-HAA-treated rats were also halted at round
spermatid, with a few reaching cap phase. The (+)-HAA-treated rats
were halted at round spermatid, with a few spermatogenic cells
showing elongation (acrosome phase). Both HAA-treated groups had
smaller Leydig cells than control and (+)-Z-BDDA-treated rats.
Conclusions
[0265] These results demonstrate that the estrogenic carboxylic
acids BDDA and HAA significantly reduce the size of the prostate in
post-pubertal male rats, and suggests their use in the treatment of
prostatic disease. This phenomenon may occur via an
estrogen-induced apoptotic mechanism. Treatment with the (+)-Z-BDDA
enantiomer resulted in a different effect from that observed with
the other estrogenic compounds in that testis size, and more
importantly spermatogenesis and Leydig cell function, was not
compromised. The other estrogens used in this study significantly
shrank the testes, and decreased its gametic and endocrine
function. As in the testes, (+)-Z-BDDA also did not significantly
shrink the seminal vesicles. The observation that (+)-Z-BDDA
shrinks the prostate without appreciably affecting the testes or
seminal vesicles is novel among estrogenic compounds, and may be
indicative of selective estrogen receptor modulation (SERM)
activity in males. SERM activity has been reported in the female,
with compounds such as tamoxifen, nafoxidine, and raloxifene, but
not in males. This differential effect of (+)-Z-BDDA also appears
to be dependent on dose, since previous studies have shown that a
dose 25 times higher (2.5 .mu.g/g bodyweight) shrank the testis,
similar to the effect of estradiol and (-)-Z-BDDA. Note Example 2,
above, and Banz, W J, T A Winters, Y-Q Hou, S R Adler, and C Y
Meyers. "Comparative Effects of the Selective Estrogen Receptor
Modulators (-)-, (+)-, and (.+-.)-Z-Bisdehydrodoisynolic Acids on
Metabolic and Reproductive Parameters in Male and Female Rats."
Horm Metab Res 30:730-736 (1998).
[0266] Since the effects of the BDDA and HAA estrogenic carboxylic
acids were observed in intact, non-castrate male rats, the present
data suggest clinical applications for these or similar compounds
in treating males with prostatic disease. These applications could
be alone or in combination with other treatments or therapies. The
(-)-Z-BDDA and both HAA enantiomers appear to be useful in treating
severe prostatic carcinoma since they cause atrophy of the
prostate, and probably decrease the androgen secretion of the
testis, which is indicative of the atrophy of the Leydig cells.
Androgens exacerbate the division and metastasis of prostatic
cancer cells. Gudziak, M R, and A Y Smith. "Hormonal Therapy for
Stage D Cancer of the Prostate" West J Med 160:351-359 (1994). The
BDDA compounds, and possibly the HAA compounds, may have advantages
over other estrogen therapies in that they also lower certain
cardiovascular risk factors. Note Example 2, above, and Banz, W J,
T A Winters, Y-Q Hou, S R Adler, and C Y Meyers. "Comparative
Effects of the Selective Estrogen Receptor Modulators (-)-, (+)-,
and (.+-.)-Z-Bisdehydrodoisynolic Acids on Metabolic and
Reproductive Parameters in Male and Female Rats." Horm Metab Res
30:730-736 (1998).. Other estrogen therapies have well-documented
cardiovascular side effects. Jacobi, G H. "Hormonal Treatment of
Metastatic Carcinoma." In: The Prostate, pp.119-128.(J M
Fitzpatrick and R J Krane, eds., Churchill Livingstone, New York,
N.Y. 1989). In addition to prostate cancer, (+)-Z-BDDA appears to
have utility in the treatment of benign prostate hypertrophy (BPH)
since the prostate is reduced without compromising spermatogenesis
and/or androgen production by the testes. In addition, even though
(+)-Z-BDDA shrank the prostate, histological analysis indicates
that the exocrine function of this accessory gland is not
appreciably compromised. The exocrine function of the seminal
vesicles with (+)-Z-BDDA is probably also unaffected. Therefore,
together with no effect on spermatogenesis, semen production should
not be affected.
[0267] Other applications of these and related estrogenic
carboxylic acids suggested by the present data include treatment of
other androgen-responsive physiological or pathological conditions,
a method of male birth control, and a means for chemical castration
in males.
Example 5
Effects of Z-Bisdehydrodoisynolic Acids on Antioxidant Capacity in
an Oxidized LDL Lag Time Assay
[0268] The effects of several synthetic and environmental
estrogens, i.e., (+)-and (-)-Z-BDDA, (+)-hydroxyvallestril
(allenolic acid) and (-)-hydroxyvallestril (allenolic acid),
genistein (soy phytoestrogen), daidzein (soy phytoestrogen),
4-hydroxy-tamoxifen, and estradiol (E2), on antioxidant capacity in
an oxidized LDL lag time assay were compared in order to assess the
antioxidant activity of these compounds.
[0269] Experiments were carried out on dialyzed LDL collected from
four fasted persons. The LDL was used within 10 days of dialysis.
The oxidizing agent was 3 .mu.M Cu.sub.2SO.sub.4; phosphate
buffered saline was used to control pH, and all drugs were
dissolved in ethanol; final concentrations of each drug in the
assays were 10.sup.-4, 10.sup.-5, 10.sup.-6, and 10.sup.-7 M. The
combined results are shown in FIG. 6.
[0270] Relative to the LDL/Cu curve, the (+)- and (-)-Z-BDDA curves
were shifted to the right and somewhat flattened (data not shown).
This shift in lag time to the right and flattening of the curves
indicates that the Z-BDDAs exhibited significant antioxidant
activity. Similar effects were also observed in the case of
(+)-allenolic acid, (-)-allenolic acid, and 4-hydroxytamoxifen.
Less antioxidant activity was observed with genistein and daidzein.
Estradiol exhibited very little antioxidant activity under these
conditions (data not shown).
[0271] The results indicate that (+)- and (-)-Z-BDDA, (+)- and
(-)-hydroxyvallestril, and 4-hydroxytamoxifen were the most potent
antioxidants.
[0272] Taken together, the results presented in Examples 2-5
suggest that the non-steroidal, estrogenically active carboxylic
acids of the present invention can be used in efficacious treatment
programs for endocrine- and non-endocrine responsive conditions in
males and females, e.g., prostatic disease, hormone-responsive
cancers, osteoporosis, therapeutic applications for pre- and
post-menopausal women, Alzheimer's disease, male pattern baldness,
and as fertility (anti-atresia) and anti-fertility agents. These
results further suggest clinical applications for the compounds
disclosed herein, as well as appropriate derivatives thereof, in
males at risk for cardiovascular disease via decreased oxidation of
LDL, for reduction of cholesterol, blood glucose, and body weight,
and to achieve positive alterations in body fat distribution. These
results also suggest methods for treating or preventing prostatic
diseases including benign prostate hyperplasia and other related
conditions, androgen-responsive pathological conditions in males,
and methods for male birth control and chemical castration,
employing estrogenic carboxylic acids.
One-Pot Asymmetric Synthesis of (+)- and
(-)-3-[2-(6-Methoxynaphthyl)]-2,2- -dimethylpentanoic Acid
Esters
[0273] In addition to compounds, compositions, and methods for
treating diseases, symptoms, and conditions responsive to the
compounds disclosed herein, the present invention also provides new
synthetic methods for preparing certain of these compounds. In
particular, the present invention provides a direct one-pot
synthesis to produce esters of
3-[2-(6-methoxynaphthyl)]-2,2-dimethylpentanoic acid (Scheme 4)
from commercially available starting material. These esters can
then be easily hydrolyzed under basic or acidic conditions to give
2 or 3. Although there are three reaction steps in this synthetic
route, separation of intermediates is unnecessary, lowering the
cost of production by saving chemicals and manpower, and increasing
product yield. 27
[0274] When a chiral R* group is used (Scheme 4), an asymmetric
induction in the Michael addition step is expected. By using
different chiral R* groups, it is possible to obtain one or the
other enantiomer directly from the reaction, eliminating the
resolution step and further lowering the cost of production. 28
[0275] This synthetic scheme can also be used to prepare compounds
having other different substituents either on the naphthalene ring
or on the propionic acid side chain, as shown in the following
structure, 6, where R can be any substituent that does not
interfere with the 10 reactions. Examples of R include, but are not
limited to, hydrogen, alkyl, alkoxy, alkylthio, alkoxyalkyl,
alkylthioalkyl, dialkylamino, halogen, aryl, aryloxy, arylthio,
alkanesulfonyl, alkanesulfinyl, silyloxy, protected ketone, and
aldehyde (e.g., ketal and acetal). 29
[0276] The major starting materials for this synthesis would have
the following structures, e.g., 7 and 8, in which X is a halogen
atom, for example Cl, Br, or I. Compound 8 is a derivative of
acrylic acid, in which Y is a heteroatom, preferably oxygen or
nitrogen. 30
[0277] The experiments described below were carried out in ethereal
solution starting from 2-bromo-6-methoxynaphthalene at pressures
ranging from 0.1 to 100 atmospheres. Other suitable solvents
include, for example, ethers, alkanes, and aromatic hydrocarbons.
The temperature can range from-100.degree. C. to .+-.150.degree. C.
Metals that can be used for these reactiond include magnesium,
lithium, sodium, potassium, calcium, palladium, copper, and
aluminum. This reaction can also be catalyzed by copper (I) halides
alone, or in the presence of other co-calalysts, such as phosphines
and boron trifluoride. Chiral auxiliary groups used to induce
asymmetric Michael addition include those derived from L- or
D-menthol, L- or D-camphor, proline-derived amines and amides. The
reaction can also be carried out in the presence of other
asymmetric compounds, such as (-)-sparteine, which can induce
asymmetric Michael additions under similar reaction conditions.
[0278] The starting materials for this synthesis can have the
structures illustrated by 7 and 8. The methyl group for the
methylation can be derived from methyl iodide, dimethyl sulphate,
methyl arenesulfonate, methyl alkanesulfonate, etc.
Example 6
Preparation of L-menthyl trans-2-methyl-2-pentenoate
[0279] To a 100-mL round-bottomed flask trans-2-methyl-2-pentenoic
acid (11.4 g, 100 mmol) and thionyl chloride (18 mL, 210 mmol) were
added. Bubbles evolved from the light-yellow solution immediately.
The mixture was stirred at room temperature for 5 min and then
heated to reflux for 30 min, during which time the mixture turned
brown. Unreacted thionyl chloride was removed by distillation.
L-menthol (15.4 g, 99 mmol) was added to the formed acyl chloride
and the mixture was heated in a 160.degree. C. oil bath of for 1
hour, at which time the evolution of HCl ceased. The mixture was
transferred into a separatory funnel and the flask rinsed with
hexanes (100 mL). The hexanes solution was then washed with aqueous
NaHCO.sub.3 solution and water. Removal of hexanes in vacuo
followed by vacuum distillation gave a light yellow oil, 18.7 g;
yield: 75%.
Example 7
Preparation of L-menthyl
3-[2-(6-Methoxynaphthyl)]-2,2-dimethylpentanoate
[0280] To a 25-mL, three-necked round-bottomed flask equipped with
a stir bar and a condenser, freshly ground magnesium turnings (0.29
g, 12.1 mmol) were added. The condenser and the flask were sealed
with rubber septa and 5 mL of freshly distilled dry THF was
injected. Argon was bubbled into the reaction flask to replace air,
followed by dropwise injection of 1,2-dibromoethane (0.2 mL, 2.3
mmol). The reaction mixture started to reflux shortly without
external heating. A solution of 2-bromo-6-methoxynaphthalene
(Aldrich, 2.37 g, 10 mmol) in dry THF was syringed dropwise into
the flask at a speed to maintain the reflux. After all the solution
was added, the mixture was heated to maintain reflux for 40 min
before being cooled in an ice-water bath. L-menthyl
trans-2-methyl-2-pentenoate (2.2 g, 8.7 mmol) was dissolved in 5 mL
of dry THF and the solution was injected into the flask. The
ice-water bath was removed and the mixture was stirred at room
temperature for 1.5 h before methyl iodide (0.62 mL, 10 mmol) was
added via a syringe. The reaction proceeded for 15 min before being
quenched with water. Product was extracted with ether and the
ethereal solution was dried over magnesium sulfate. Removal of
ether in vacuo gave a light yellow oil, 3.33 g. Column
chromatography (silica gel, hexanes:ethyl acetate=50:1) provided a
yellow oil, 2.85 g, whose .sup.1H NMR spectrum showed the presence
of L-menthyl 3-[2-(6-methoxynaphthyl)]-2,2-dimethylpentanoate as
the major product. Yield by NMR: 77%. The two diastereomers exist
in equal amount.
Example 8
Preparation of (-)-8-phenylmethyl trans-2-methyl-2-pentenoate
[0281] To a 50-mL round-bottomed flask trans-2-methyl-2-pentenoic
acid (2.2 g, 19.3 mmol) and thionyl chloride (5 mL, 58 mmol) were
added. Bubbles evolved from the light yellow solution immediately.
The mixture was stirred at room temperature for 5 min and then
heated at reflux for 30 min. Unreacted thionyl chloride was removed
by heating the mixture in an oil bath at 160.degree. C.
(-)-8-Phenylmenthol (Aldrich, 0.95 g, 4.1 mmol) was added to the
acyl chloride and the mixture was heated in an oil bath at
190.degree. C. for 30 min. Dilute aqueous KOH solution was added
into the mixture and the product was extracted with ether.
Evaporation of ether provided a light brown oil which is further
purified by column chromatography (silica gel, hexanes:ether=50:1)
to give a light yellow oil, 1.28 g; yield: 94.8%. .sup.1H NMR
showed that it was the desired product, but contained some cis
isomer, the trans:cis ratio being 5:1.
Example 9
Preparation of (-)-8-phenylmethyl
3-[2-(6-Methoxynaphthyl)]-2.2-dimethyl-p- entanoate
[0282] To a 25-mL, three-necked round-bottomed flask equipped with
a stir bar and a condenser, freshly ground magnesium turnings
(0.171 g, 7.1 mmol) were added. The condenser and the flask were
sealed with rubber septa and 5 mL of freshly distilled dry THF was
injected. Argon was bubbled into the reaction flask to replace air,
followed by dropwise injection of 1,2-dibromoethane (0.18 mL, 2.1
mmol). The reaction mixture started to reflux shortly without
external heating. A solution of 2-bromo-6-methoxynaphthalene (1.20
g, 5.1 mmol) in dry THF (10 mL) was syringed dropwise into the
flask at a speed to maintain the reflux. After all the solution was
added, the mixture was heated to maintain reflux for 45 min before
being cooled in an ice-water bath. (-)-8-Phenylmenthyl
trans-2-methyl-2-pentenoate (1.27 g, 3.8 mmol) was dissolved in 5
mL of dry THF and the solution was injected into the flask. The
ice-water bath was removed and the mixture was stirred at room
temperature for 1.5 h before methyl iodide (0.4 mL, 6.4 mmol) was
syringed into the flask. After 15 min the reaction was quenched
with aqueous NH.sub.4Cl solution. The product was extracted with
ether, the ethereal solution was dried over magnesium sulfate, and
the ether was removed in vacuo to yield a light-yellow thick oil.
Column chromatography (silica gel, hexanes:ethyl
acetate=50:1.about.20:1) provided a yellow oil, 1.04 g, whose
.sup.1H NMR spectrum showed that the two diastereomers exist in a
ratio of about. 1.7:1. Yield: 77% based on reacted starting
material.
[0283] The present invention thus provides direct, one-pot methods
for the asymmetric synthesis of esters of (+)- and
(-)-3-[2-(6-methoxynaphthyl)]-- 2,2-dimethylpentanoic acid and
esters of other substituted 3-(2-naphthyl)propionic acids. These
esters can be easily hydrolyzed into their corresponding free
acids.
[0284] The invention being thus described, it will be obvious that
the same can be varied in many ways. Such variations are not to be
regarded as a departure from the spirit and scope of the present
invention, and all such modifications and equivalents as would be
obvious to one skilled in the art are intended to be included
within the scope of the following claims.
[0285] The contents of each of the references cited herein are
hereby incorporated by reference in their entirety.
* * * * *