U.S. patent application number 11/682072 was filed with the patent office on 2007-09-13 for prodrugs for use as ophthalmic agents.
Invention is credited to Katalin Prokai, Laszlo Prokai.
Application Number | 20070213310 11/682072 |
Document ID | / |
Family ID | 38225311 |
Filed Date | 2007-09-13 |
United States Patent
Application |
20070213310 |
Kind Code |
A1 |
Prokai; Laszlo ; et
al. |
September 13, 2007 |
Prodrugs for Use as Ophthalmic Agents
Abstract
The subject invention provides a mechanism by which steroidal
quinol compounds confer beneficial ophthalmic effects. The subject
compounds possess a lipophilic-hydrophilic balance for transcorneal
penetration and are readily reduced into parent phenolic A-ring
steroid compounds to provide protection or treatment against
various ocular symptoms and disorders. The compounds according to
the subject invention appear to be highly advantageous as prodrugs
to provide protection and/or treatment against ocular disorders.
These prodrugs confer lipid solubility optimal for transocorneal
penetration and are readily converted to endogenous reducing agents
into active phenolic A-ring steroid compounds. To the extent that
these prodrugs have reduced feminizing effects and systemic
toxicity, they would be expected to be quite advantageous for
protecting or treating the eye against ocular disorders such as
cataract or glaucoma without undesired (systemic) side
effects).
Inventors: |
Prokai; Laszlo; (Mansfield,
TX) ; Prokai; Katalin; (Mansfield, TX) |
Correspondence
Address: |
SALIWANCHIK LLOYD & SALIWANCHIK;A PROFESSIONAL ASSOCIATION
PO BOX 142950
GAINESVILLE
FL
32614-2950
US
|
Family ID: |
38225311 |
Appl. No.: |
11/682072 |
Filed: |
March 5, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10731528 |
Dec 9, 2003 |
7186707 |
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11682072 |
Mar 5, 2007 |
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10405413 |
Apr 1, 2003 |
7026306 |
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10731528 |
Dec 9, 2003 |
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60369589 |
Apr 1, 2002 |
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60432354 |
Dec 9, 2002 |
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Current U.S.
Class: |
514/178 ;
514/182; 552/612 |
Current CPC
Class: |
A61K 31/57 20130101;
C07J 1/00 20130101 |
Class at
Publication: |
514/178 ;
514/182; 552/612 |
International
Class: |
A61K 31/56 20060101
A61K031/56; C07J 51/00 20060101 C07J051/00 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with government support under a
grant awarded from the National Institute of Neurological Disorders
and Stroke under grant number NS44765, and a grant from the
National Institutes of Health on Aging under grant number PO1
AG10485. The government has certain rights in the invention.
Claims
1. A method for treating a patient diagnosed with at least one
ophthalmic disorder, wherein said method comprises administering to
the patient an effective amount of a steroidal quinol, wherein the
steroidal quinol is converted to biologically active
2-(1-adamantyl)-estra-1,3,5(10)-triene-3,17.beta.-diol in vivo.
2. The method of claim 1, wherein the steroidal quinol is
2-(1-adamantyl)-10.beta.,17.beta.-dihydroxyestra-1,4-dien-3-one.
3. The method, according to claim 1, further comprising
administering the quinol by a route selected from the group
consisting of oral, buccal, intramuscular, transdernal,
intravenous, and subcutaneous.
4. The method, according to claim 1, wherein the ophthalmic
disorders are selected from the group consisting of retinitis
pigmentosa, conjunctivitis, diabetic retinopathy, dry eye, macular
degeneration, glaucoma, and cataracts.
5. A quinol that is converted to a biologically active
2-(1-adamantyl)-estra-1,3,5(10)-triene-3,17.beta.-diol and having
the structure ##STR13##
6. A biologically active compound having the structure
##STR14##
7. A pharmaceutical composition comprising a quinol that is
converted to a biologically active estrogen compound via enzyme
catalyzed reduction, wherein said composition further comprises a
pharmaceutically acceptable carrier, wherein the quinol has the
structure: ##STR15##
8. A pharmaceutical composition comprising a biologically active
estrogen compound having the structure: ##STR16##
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of co-pending
application Ser. No. 10/731,528, filed Dec. 9, 2003; which is a
continuation-in-part application of U.S. Ser. No. 10/405,413, filed
Apr. 1, 2003; which claims the benefit of U.S. provisional patent
application Ser. No. 60/369,589, filed Apr. 1, 2002. This
application also claims the benefit of U.S. provisional patent
application Ser. No. 60/432,354, filed Dec. 9, 2002. These
applications are hereby incorporated by reference in their
entirety, including all figures and tables.
FIELD OF THE INVENTION
[0003] The present invention relates to prodrugs for use as
ophthalmic agents, specifically for retinal protection. In
particular, the present invention relates to the use of steroidal
quinols as prodrugs of phenolic A-ring steroid compounds to treat
and/or prevent eye pathologies.
BACKGROUND OF INVENTION
[0004] A variety of tissues metabolize estrogen (as a
representative phenolic A-ring steroid) to various degrees. Of all
of the tissues investigated, cornea appears to be the most active
estrogen-metabolizing tissue (Starka, L and J Obenberger, "In vitro
Estrone-Estradiol-17.beta. Interconversion in the Cornea, Lens,
Iris and Retina of the Rabbit Eye," Arch Klin Exp Ophthalmol,
196:199-204 (1975)). Estrogens have demonstrated an important role
in the health maintenance of all mucous membranes in the body,
including the maintenance of a healthy ocular surface. Additional
studies have revealed that the biological activity of estrogen may
be effective in the protection and treatment of the eye, including
the lens and retina, against cataracts and the detrimental effects
of glaucoma.
[0005] Unfortunately, many regions of the eye are relatively
inaccessible to systemically administered estrogens. For example,
orally administered estrogen passes through the liver before
reaching estrogen sensitive tissues. Because the liver contains
enzymes that can inactivate the estrogen, the estrogen that
eventually reaches tissue targeted for treatment is virtually
ineffective. Moreover, systemic administration of estrogen often
produces undesirable side effects, i.e., feminizing side effects in
men.
[0006] As a result, topical drug delivery remains the preferred
route of administration to the eye. There are a variety of factors
that affect the absorption of drugs into the eye. These factors
include: the instillation volume of the drug, the frequency of
instilled drug administration, the structure and integrity of the
cornea, the protein level in tears, the level of enzymes in tears,
lacrimal drainage and tear turnover rate, as well the rate of
adsorption and absorption of a drug by the conjunctiva, sclera, and
eyelids.
[0007] Thus, the potential treatment of ocular disorders/conditions
by estrogens or agents derived from estrogens is confounded by poor
ocular bioavailability of pharmacologically active agents and by
the likelihood of triggering systemic side effects associated with
the administration of natural (endogenous) estrogens. The latter
are due to absorption from the nasal cavity and the
gastrointestinal (GI) tract after the topically administered
estrogen hormone gains access to these pathways through its removal
by the nasolacrimal apparatus of the eye. A potential way of
reducing or even eliminating systemic side effects is to improve
ocular targeting that would allow for the use of reduced doses of
the biologically active agent in the ophthalmic drug formation.
[0008] Accordingly, the direct administration to an eye lens of
estrogen having quinolines (i.e., 6-hydroxyquinoline) and fused
quinolines that act as steroid receptor modulators to prevent or
treat cataract disorders has been disclosed. In addition, the
administration of 17-.beta.-estradiol to the surface of the eye to
alleviate dry-eye syndrome or keratoconjunctivitis sicca has been
disclosed. Glycosides of catechol estrogens have been formulated
that demonstrate antioxidant activity to the same degree as to that
of the parent catechol estrogens. Nonetheless, all of the
previously disclosed compounds and methods for applying estrogens
to the eye relate to compounds that lack efficient corneal
penetration and/or are unapplicable to men because of their
activity as a female hormone.
[0009] As noted above, the major barrier to ocular drug penetration
is the cornea. The cornea is composed of three layers: a lipid-rich
epithelium, a lipid-poor soma, and a lipid-rich endothelium.
Therefore, an agent must possess both lipophilic-hydrophilic
balance for adequate transcorneal penetration and, thus, ocular
bioavailability (Akers H J, "Ocular bioavailability of topically
applied ophthalmic drugs," Am Pharm, NS23:33-36 (1983)). Thus, poor
ocular bioavailability is an issue for estrogens and their
synthetic analogs, because estrogens are highly lipid soluble
molecules that are usually not amenable to adequate transcorneal
penetration.
[0010] Prodrugs are inactive compounds that are converted in vivo
into biologically active agents by enzymatic and/or chemical
transformations. Prodrugs are advantageous because they can be
designed to overcome problems associated with stability, toxicity,
lack of specificity, or limited bioavailability, that may exist
with the active form of a drug. Thus, there is a need to develop
effective prodrugs of estrogen as a medical compound.
[0011] Estrogen quinols have been known for decades among organic
chemists (Gold A. M., and Schwenk E., "Synthesis and reaction of
steroidal quinols," J Am Chem Soc, 80:5683-5687 (1958)) though
their metabolic formation has only been reported recently (Ohe T.,
et al., "Novel metabolic pathway of estrone and 17.beta.-estradiol
catalyzed by cytochrome P-450", Drug Metab Dispos, 28:11-112
(2000)). 10.beta.-hydroxy-1,4-estradiene-3,7-dione and
10.beta.,17.beta.-dihydroxy-1,4-estradiene-3-one were detected from
the respective estrogens during metabolic oxidation catalyzed by
several cytochrome P-450 isoenzymes in rat liver microsomal
systems. Contrary to well-known catechol metabolites of estrogens
(Zhu, B. T. and Conney A. H., "Functional role of estrogen
metabolism in target cells: review and perspective,"
Carcinogenesis, 19:1-27 (1998)), quinols do not possess an aromatic
A-ring, making their biochemistry substantially different from that
of catechols. Studies are currently underway to assess the nature
of estrogen quinols.
BRIEF SUMMARY
[0012] The subject invention provides materials and methods wherein
unique and advantageous steroidal quinols are used for a broad
range of therapeutic purposes, including the treatment or
prevention of ophthalmic disorders and/or conditions by modulating
or activating estrogen receptors. These disorders and/or conditions
include, but are not limited to, conjtnctivitis, diabetic
retinopathy, dry eye, glaucoma, and cataract.
[0013] A quinol (i.e., the
10.alpha.,.beta.-hydroxyestra-1,4-diene-3-one structures) derived
synthetically from phenolic A-ring steroids has been found to
confer significant reduced lipid solubility compared to the parent
phenolic A-ring steroid compounds to provide improved transcorneal
penetration. Further, these quinols can be converted to phenolic
A-ring steroid structures by endogeneous NAD(P)H as a reducing
agent. In one embodiment, an oxidoreductase catalyst converts
subject steroidal quinols to phenolic A-ring steroids that possess
pharmacological activity in the eye. The present invention exploits
the benefits of prodrugs (including but not exclusively based on
the quinol structure as a novel pro-moiety) for phenolic A-ring
steroid compounds to provide ocular bioavailability of the
therapeutic agent in question. Prodrugs are, by definition,
inactive compounds that are converted to the biologically active
agents by chemical or enzymatic transformation in vivo.
[0014] The subject invention provides a mechanism by which quinol
derived phenolic A-ring steroid compounds confer beneficial
ophthalmic effects. The subject compounds possess a
lipophilic-hydrophilic balance for transcorneal penetration and are
readily reduced into parent phenolic A-ring steroid compounds to
provide protection or treatment against various ocular symptoms and
disorders. The compounds according to the subject invention appear
to be highly advantageous as prodrugs to provide protection and/or
treatment against ocular disorders. These prodrugs confer low lipid
solubility and are readily converted by endogenous reducing agents
into active phenolic A-ring steroid compounds. To the extent that
these prodrugs have reduced feminizing effects and systemic
toxicity, they would be expected to be quite advantageous for
protecting or treating the eye against ocular disorders such as
cataract or glaucoma.
[0015] In a specific embodiment, the subject invention provides
steroidal quinol compounds that are, themselves, inactive. However,
these quinol structures can act as prodrugs because they are
converted to a therapeutically active phenolic A-ring steroid upon
exposure to a reducing agent. Additionally, because an active
phenolic A-ring steroid compound arises after conversion by a
reducing agent, a smaller concentration of the steroidal quinols is
required as compared to direct administration of phenolic A-ring
steroid, thus reducing the potential for systemic toxicity.
[0016] In one particular embodiment of the subject invention,
isomers of 10-hydroxyestra-1,4-diene-3-one quinol structure
(estrone-quinol) are converted to active, phenolic A-ring steroid
compounds (i.e., estrone) when exposed to a reducing agent. In
related embodiments, quinols are derived from estrogen analogues,
i.e., 3,17-dihydroxyestra-1,3,5(10),9(11)-tetraene (ZYC1);
2-(1-adamantyl)-estra-1,3,5(10)-triene-3,17.beta.-diol; and
2-(1-adamantyl)-10.beta.,17.beta.-dihydroxyestra-1,4-dien-3-one.
[0017] In another embodiment, steroidal quinols are provided as
prodrugs that require at least one-step activation in vivo to yield
pharmaceutically active estrogen compounds. In a related
embodiment, quinols derived from estrogen prodrugs that require
two-step activation can include a polar functional group to enhance
hydrophilicity at the 17-OH group or may have the 10-OH group
esterified to decrease lipophilicity through phosphate, or
N,N,N-trialkylammonium esters.
[0018] In another embodiment, the 3,17-keto groups of quinols of
the present invention can be functionalized as oxime and/or
alkoximes. In doing so, preliminary compounds to the subject
quinols are created (to form i.e. pro-prodrugs). Such
functionalized quinols (i.e., 3-keto functionalized as an oxime)
can be used for a variety of therapeutic purposes, including use
for ocular-specific delivery of phenolic A-ring steroids.
[0019] An object of the present invention is to provide compounds
formulated for ophthalmic administration. For example, solutions or
suspensions of these compounds may be formulated in the form of eye
drops, or membranous ocular patch, which is applied directly to the
surface of the eye.
[0020] It is another object of the subject invention to provide an
ophthalmic agent with an increased therapeutic index associated
with treatments using the subject compounds disclosed herein.
BRIEF DESCRIPTION OF THE FIGURES
[0021] FIG. 1 illustrates the viability of retinal ganglial cells
in the presence of glutamate, estrogen analog
3,17-dihydroxyestra-1,3,5(10),9(11)-tetraene (ZYC1), or
combinations of glutamate and various concentrations of ZYC1.
[0022] FIG. 2 illustrates retinal ganglial cell viability when
treated with glutamate in the presence or absence of ZYC1 or ZYC1
incubated in the presence of various concentrations of estrogen
receptor antagonist, ICII82,780 (ICI).
[0023] FIG. 3 illustrates a quinol acetate in accordance with the
subject invention.
[0024] FIG. 4 illustrates an (alk)oxime of a quinol, in accordance
with the subject invention.
[0025] FIG. 5 illustrates the viability of retinal ganglial cells
in the presence of glutamate, phenolic A-ring steroid
2-(1-adamantyl)-3-hydroxyestra-1,3,5(10)-trien-17-one (ZYC3), or
combinations of glutamate and various concentrations of ZYC3.
[0026] FIG. 6 illustrates LC/MS/MS analysis demonstrating the
detection of 10.beta.-hydroxyestra-1,4-dien-3,17-dione
(estrone-quinol, t.sub.R=1.38) and a product formed from it
(t.sub.R=4.5 min.) after the incubation of estrone-quinol with
NADPH.
[0027] FIG. 7 illustrates MS/MS analysis of the chromatographic
peak at t.sub.R=4.5 min., which is identical to that of
estrone.
[0028] FIG. 8 illustrates MS.sup.3 recording from the
chromatographic peak, t.sub.R=4.5 min., m/z 253 selected as
precursor after MS/MS, which is identical to that of estrone.
[0029] FIG. 9 illustrates the efficacy of both the prodrug
2-(1-adamantyl)-.DELTA..sup.1-dehydro-19-nortestosterone and the
active agent 2-(1-adamantyl)-estra-1,3,5(10)-triene-3,17.beta.-diol
in protecting the outer nuclear layer (ONL) of the retina in
heterozygous S334ter rhodopsin mutation transgenic rats.
DETAILED DISCLOSURE
[0030] The subject invention provides steroidal quinol compounds
that produce phenolic A-ring steroids in vivo. In one embodiment,
these compounds provide improved physicochemical properties
including, but not limited to, favorable ocular bioavailability and
facile transcorneal penetration. In a preferred embodiment,
estrogen derived quinol compounds demonstrate decreased
lipophilicity as compared to lipophilic estrogens and estrogen
analogues.
[0031] In another embodiment of this invention, these compounds
treat and/or protect against various ocular diseases. Preferred
compounds of the subject invention are effective in treating and/or
preventing maladies associated with vision-threatening intraocular
damage due to pathophysiological predispositions. Particularly
preferred compounds are those which treat glaucoma and/or macular
degeneration.
[0032] In a specific embodiment, the subject invention provides
steroidal quinol compounds that are, themselves, inactive. However,
these quinol structures can act as prodrugs because they are
converted to a therapeutically active phenolic A-ring steroid upon
exposure to a reducing agent. Additionally, because an active
phenolic A-ring steroid compound arises after conversion by a
reducing agent, a smaller concentration of the steroidal quinols is
required due to their improved ocular bioavailability as compared
to direct administration of estrogen, thus reducing the potential
for systemic toxicity. In a related embodiment of the subject
invention, steroidal quinols are provided as prodrugs that are
converted into an active phenolic A-ring steroid via a one-step
conversion by a reducing agent. Suitable reducing agents include
endogenous NAD(P)H or oxidoreductases.
[0033] In one particular embodiment of the subject invention, a
10.beta.-hydroxyestra-1,4-diene-3-one quinol structure
(estrone-quinol) is converted to an active, phenolic A-ring
estrogen compound (estrone) when exposed to a reducing agent. In
related embodiments, quinols are derived from estrogen analogues,
i.e., 3,17-dihydroxyestra-1,3,5(10),9(11)-tetraene (ZYC1);
2-(1-Adamantyl)estrone (ZYC3);
2-(1-Adamantyl)-estra-1,3,5(10)-triene-3,17.beta.-diol; and
2-(1-Adamantyl)-10.beta.,17.beta.-dihydroxyestra-1,4-dien-3-one.
[0034] In another embodiment, steroidal quinols are provided as
prodrugs that require two (or more than two) step activation in
vivo to yield pharmaceutically active estrogen compounds. The
liberation of a parent estrogen occurs through a two-step reaction:
(1) enzymatic (phosphatase, esterase) cleavage of the ester group
followed by (2) spontaneous and fast chemical conversion of a
quinol by an endogenous reducing agent. In a related embodiment,
these compounds according to the present invention can include a
polar functional group to enhance hydrophilicity at the 17-OH group
or may have the 10-OH group esterified to decrease lipophilicity
through phosphate or N,N,N-trialkylammonium esters (such as
2-(1-adamantyl)-estra-1,3,5(10)-triene-3,17.beta.-diol and
2-(1-adamantyl)-10.beta.,17.beta.-dihydroxyestra-1,4-dien-3-one).
[0035] In another embodiment, the prodrugs according to the subject
invention can be synthesized by attaching a polar functional group
to enhance affinity to water and facilitate the transport of the
prodrug of the subject invention through the lipid-poor middle
stroma in the cornea. In a preferred embodiment, the 17-OH group of
a quinol according to the subject invention is the primary site to
which a polar functional group is added. In another preferred
embodiment, the 10.beta.-OH of a steroidal quinol (i.e.,
17-hydroxyestra-1,4-diene-17-one) is blocked by esterification to
make the resultant prodrug more lipophilic than the phenolic A-ring
steroid derived quinol.
[0036] It will be noted that the structure of some of the compounds
of this invention includes asymmetric carbon atoms. It is to be
understood accordingly that the isomers arising from such asymmetry
(i.e., all enantiomers and diastereomers) are included within the
scope of this invention, unless indicated otherwise. Such isomers
can be obtained in substantially pure form by conventional methods
including, for example, by classical separation techniques and by
stereochemically controlled synthesis.
Definitions
[0037] A number of terms are used herein to designate particular
elements of the present invention. When so used, the following
meanings are intended:
[0038] The term "estrogen," as used herein, refers to both
naturally occurring and synthetic substances classed as estrogen on
the basis of their therapeutic or biological action (see listing
under `Estrogens` in the `Therapeutic Category and Biological
Activity Index` of The Merck Index, 12th Edition, Merck Research
Laboratories, NJ, 1996, page THER-22). According to this listing,
estrogens may be steroids (i.e., estradiol, ethinyl estradiol,
colpormon, conjugated estrogenic hormones, equilenin, equilin,
estriol, estrone, mestranol, moxestrol, mytatrienediol,
quinestradiol and quinestrol) or non-steroids (i.e.,
diethylstilbestrol, dienestrol, benzestrol, broparoestrol,
chlorotrianisene, dimestrol, fosfestrol, hexestrol,
methallenestril, methestrol). Additional substances known to be
estrogenic, that is, they interact with cellular estrogen receptors
and mimic the effects of estrogens, include estrogenic substances
that have been shown to be tissue selective in their estrogenic
effects. Diverse classes of molecules fall within this category,
for example: quinolines and fused quinolines that act as steroid
receptor modulators such as
3,9-dihydroxy-5H-benzofuro[3,2-c]quinoline-6-one and those
disclosed in WO 96/19458; phytoestrogens which occur naturally in
plants such as forage plants, soya beans, seeds, berries and nuts
(Jordan et al., "Structure-activity relationships of estrogen,"
Env. Health Per., 61:97-110 (1985)), including isoflavones such as
genistein and genistein glycosides, equol, O-desmethyl-angolensin,
biochanin A, daidzein and formononetin; flavones such as phloretin,
4'-6-dihydroxyflavone and tricin, and coumestans such as
coumestrol, 4'-O-methyl coumestrol, medicagol and sativol, lignans
such as matairesinol, enterodiol, enterolactone, guaiaretic acid,
nordihydroguaiaretic acid and derivatives thereof,
.beta.-sitosterol; mycoestrogens such as zeranol, zearalenol and
zearalenone; estrogen receptor agonist/antagonists, such as
tamoxifen, hydroxytamoxifen, zindoxifene and its metabolites,
nafoxidene and derivatives, clomiphene, centchroman,
benzothiophenes and related compounds such as
benzothiophene-derived LY139478 (Eli Lilly), raloxifene and
droloxifene, which may mimic the action of estrogens in certain
types of cells, while opposing it in others (Raisz, L. G.,
"Estrogen and bone: new pieces to the puzzle," Nature. Med.,
2(10):1077-8 (1996)); and many para-substituted phenols that
contain a strategically located phenolic hydroxyl not impaired by
an alkyl substitution (see Jordan et al., "Structure-activity
relationships of estrogen," Env. Health Per., 61:97-110 (1985)),
including octyl phenyl, nonyl phenol, butylated hydroxyanisole,
bisphenol A and trihydroxy-8-prenylflavone. Note that estrogenic
substances in this general category may also be referred to in the
literature as `estrogens` (see Jordan et al., 1985, for example).
As already described above (for `estrogens` as defined in Merck),
estrogenic substances may exert their estrogenic effect(s) directly
or they may require metabolic conversion to an active form after
administration. For example, metabolic activation of some
phytoestrogens involves demethylation to phenols (Jordan et al.,
"Structure-activity relationships of estrogen," Env. Health Per.,
61:97-110 (1985)).
[0039] The term "estrogen derived quinols," (i.e.,
10.alpha./.beta.-hydroxyestra-1,4-diene-3-one structure) as used
herein, refers to quinols and quinol derivatives related to
estrogens, as described above, and para-substituted phenols
obtained by oxidation of the phenolic ring, as described below.
[0040] The term "phenolic A-ring steroid" used herein refers to
compounds containing a 3-hydroxy-1,3,5(10)-triene moiety as the
six-membered A-ring of a steroid, steroid analogue or steroid
mimic, including compounds that manifest affinity to estrogen
receptors (i.e., 3,17-dihydroxyestra-1,3,5(10),9(11)-tetracne) as
well as compounds that do not bind to such receptors (i.e.,
2-(1-adamantyl)-3-hydroxyestra-1,3,5(10)-trien-17-one).
[0041] The term "steroidal quinol" used herein refers to a steroid
containing a 10.alpha./.beta.-hydroxy-1,4-diene-3-one moiety as the
six-membered A-ring of a steroid, steroid analogue or steroid
mimic.
[0042] The term "ophthalmic disorders," and/or "ophthalmic
conditions," as used herein, refers to ophthalmic diseases,
conditions, and/or disorders including, without limitation, those
associated with the anterior chamber of the eye (i.e., hyphema,
synechia); the choroid (i.e., choroidal detachment, choroidal
melanoma, multifocal choroidopathy syndromes); the conjunctiva
(i.e., conjunctivitis, cicatricial pemphigoid, filtering Bleb
complications, conjunctival melanoma, Pharyngoconjunctival Fever,
pterygium, conjunctival squamous cell carcinoma); connective tissue
disorders (i.e., ankylosing spondylitis, pseudoxanthoma elasticum,
corneal abrasion or edema, limbal dermoid, crystalline dystrophy
keratits, keratoconjunctivitis, keratoconus, keratopathy,
megalocornea, corneal ulcer); dermatologic disorders (i.e.,
ecrodermatitis enteropathica, atopic dermatitis, ocular rosacea,
psoriasis, Stevens-Johnson syndrome); endocrine disorders (i.e.,
pituitary apoplexy); extraocular disorders (i.e., Abducens Nerve
Palsy, Brown syndrome, Duane syndrome, esotropia, exotropia,
oculomotor nerve palsy); genetic disorders (i.e., albinism, Down
syndrome, Peters Anomaly); the globe (i.e., anophthalmos,
endophthalmitis); hematologic and cardiovascular disorders (i.e.,
Giant Cell Arteritis, hypertension, leukemias, Ocular Ischemic
syndrome, sickle cell disease); infectious diseases (i.e.,
actinomycosis, botulism, HIV, diphtheria, Escherichia coli,
Tuberculosis, ocular manifestations of syphilis); intraocular
pressure (i.e., glaucoma, ocular hypotony, Posner-Schlossman
syndrome), the iris and ciliary body (i.e., aniridia, iris prolaps,
juvenile xanthogranuloma, ciliary body melanoma, iris melanoma,
uveitis); the lacrimal system (i.e., alacrima, Dry Eye syndrome,
lacrimal gland tumors); the lens (i.e., cataract, ectopia lentis,
intraocular lens decentration or dislocation); the lid (i.e.,
blepharitis, dermatochalasis, distichiasis, ectropion, eyelid
coloboma, Floppy Eye syndrome, trichiasis, xanthelasma); metabolic
disorders (i.e., gout, hyperlipoproteinemia, Oculocerebrorenal
syndrome); neurologic disorders (i.e., Bell Palsy, diplopia,
multiple sclerosis); general ophthalmologic (i.e., red eye,
cataracts, macular degeneration, red eye, macular degeneration);
the optic nerve (i.e., miningioma, optic neuritis, optic
neuropathy, papilledema); the orbit (i.e., orbital cellulits,
orbital dermoid, orbital tumors); phakomatoses (i.e.,
ataxia-telangiectasia, neurofibromatosis-1); presbyopia; the pupil
(i.e., anisocoria, Homer syndrome); refractive disorders (i.e.,
astigmatism, hyperopia, myopia); the retina (i.e., Coats disease,
Eales disease, macular edema, retinitis, retinopathy); and the
sclera (i.e., episcleritis, scleritis).
[0043] The term "patient," as used herein, describes an organism,
including mammals, to which treatment with the compositions
according to the present invention is provided. Mammalian species
that benefit from the disclosed methods of treatment include, and
are not limited to, apes, chimpanzees, orangutans, humans, monkeys;
and domesticated animals (i.e., pets) such as dogs, cats, mice,
rats, guinea pigs, and hamsters.
[0044] The term "polar aprotic solvent" refers to polar organic
solvents lacking an easily removed proton, including, but not
limited to, ethyl acetate, dimethylformamide (DMF), and
acetonitrile.
[0045] The term "pharmaceutically acceptable salts," as used
herein, refers to those carboxylate salts, esters, and prodrugs of
the compound of the present invention which are, within the scope
of sound medical judgment, suitable for use in contact with the
tissues of humans and lower animals with undue toxicity,
irritation, allergic response, and the like, commensurate with a
reasonable benefit/risk ratio, and effective for their intended
use, as well as the zwitterionic forms, where possible, of the
compounds of the invention.
[0046] Pharmaceutically acceptable salts are well known in the art
and refer to the relatively non-toxic, inorganic and organic acid
addition salts of the compound of the present invention. For
example, S. M. Berge, et al. describe pharmaceutically acceptable
salts in detail in J. Pharmaceutical Sciences, 66:1-19 (1977) which
is incorporated herein by reference. The salts can be prepared in
situ during the final isolation and purification of the compounds
of the invention, or separately by reacting the free base function
with a suitable organic acid. Examples of pharmaceutically
acceptable, nontoxic acid addition salts are salts of an amino
group formed with inorganic acids such as hydrochloric acid,
hydrobromic acid, phosphoric acid, sulfuric acid and perchloric
acid or with organic acids such as acetic acid, oxalic acid, maleic
acid, tartaric acid, citric acid, succinic acid or malonic acid or
by using other methods used in the art such as ion exchange. Other
pharmaceutically acceptable salts include adipate, alginate,
ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,
borate, butyrate, camphorate, camphorsulfonate, citrate,
cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, formate, fumarate, glucoheptonate,
glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate,
hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate,
laurate, lauryl sulfate, malate, maleate, malonate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,
oleate, oxalate, palmitate, palmoate, pectinate, persulfate,
3-phenylpropionate, phosphate, picrate, pivalate, propionate,
stearate, succinate, sulfate, tartrate, thiocyanate,
p-toluenesulfonate, undecanoate, valerate salts, and the like.
Representative alkali or alkaline earth metal salts include sodium,
lithium, potassium, calcium, magnesium, and the like. Further
pharmaceutically acceptable salts include, when appropriate,
nontoxic ammonium, quaternary ammonium, and amine cations formed
using counterions such as halide, hydroxide, carboxylate, sulfate,
phosphate, nitrate, loweralkyl sulfonate and aryl sulfonate.
[0047] The term "pharmaceutically acceptable prodrugs," as used
herein, refers to those prodrugs of the compounds of the present
invention which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of humans and lower
animals without undue toxicity, irritation, allergic response, and
the like, commensurate with a reasonable benefit/risk ratio, and
effective for their intended use, as well as the zwitterionic
forms, where possible, of the compounds of the invention.
[0048] The term "prodrug," as used herein, refers to a derivative
of a biologically active compound (i.e., the steroidal quinols
according to the present invention) that lacks pharmaceutical
activity, but is converted (i.e., by NAD(P)H) to an active agent,
which is a phenolic A-ring steroid such as estrogen honnone,
estrogen analogue, substituted estrogen or estrogen-receptor
agonist or antagonist) upon interaction with a biological or
chemical system, for example catalyzed reduction by enzymes in the
eye. A thorough discussion is provided in T. Higuchi and V. Stella,
Pro-drugs as Novel Delivery Systems, Vol. 14 of the A.C.S.
Symposium Series, and in Edward B. Roche, ed., Bioreversible
Carriers in Drug Design, American Pharmaceutical Association and
Pergamon Press, 1987, both of which are incorporated herein by
reference. A prodrug, according to the present invention, can be
converted into an active compound with one or more steps.
[0049] The term "substituted" shall be deemed to include multiple
degrees of substitution by a named substituent. Where multiple
substituent moieties are disclosed, the substituted compound can be
independently substituted by one or more of the disclosed or
claimed substituent moieties, singly or severally.
[0050] Unless otherwise specified, as used herein, the term "alkyl"
refers to a straight or branched or cyclic alkyl moiety. In one
embodiment, the alkyl moiety is C.sub.1-20 alkyl, which refers to
an alkyl moiety having from one to twenty carbon atoms, including
for example, methyl, ethyl, propyl, isopropyl, butyl, tert-butyl,
pentyl, hexyl and octyl, cycloalkyl including for example
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl. The alkyl group
specifically includes fluorinated alkyls such as CF.sub.3 and other
halogenated aikyls such as CH.sub.2CF.sub.2, CF.sub.2CF.sub.3, the
chloro analogs, and the like. The alkyl group can be optionally
substituted with one or more moieties selected from the group
consisting of aryl, heteroaryl, heterocyclic, carbocycle, alkoxy,
heterocycloxy, heterocylalkoxy, aryloxy; arylalkoxy; heteroaryloxy;
heteroarylalkoxy, carbohydrate, amino acid, amino acid esters,
amino acid amides, alditol, halo, haloalkyl, hydroxyl, carboxyl,
acyl, acyloxy, amino, amido, alkylamino, dialkylamino, arylamino,
nitro, cyano, thiol, imide, sulfonic acid, sulfate, sulfonyl,
sulfanyl, sulfinyl, sulfamoyl, carboxylic ester, carboxylic acid,
amide, phosphonyl, phosphinyl, phosphoryl, thioester, thioether,
oxime, hydrazine, carbamate, phosphonic acid, phosphate,
phosphonate, phosphinate, sulfonamido, carboxamido, hydroxamic
acid, sulfonylimide, substituted or unsubstituted urea connected
through nitrogen; or any other desired functional group that does
not inhibit the pharmacological activity of this compound, either
unprotected, or protected as necessary, as known to those skilled
in the art, for example, as taught in Greene, et al., Protective
Groups in Organic Synthesis, John Wiley and Sons, Second Edition,
1991, hereby incorporated by reference.
[0051] The term "alkenyl" refers to a straight or branched alkyl
moiety having one or more carbon double bonds, of either E or Z
stereochemistry where applicable. This term includes for example,
vinyl, 1-propenyl, 1- and 2-butenyl, and 2-methyl-2-propenyl, as
well as "cycloalkenyl" groups such as cyclopentenyl and
cyclohexenyl.
[0052] The term "alkoxy," as used herein, and unless otherwise
specified, refers to a moiety of the structure --O-alkyl, wherein
alkyl is as defined above. The alkyl group can be optionally
substituted as described above. Alkoxy groups can include
OCF.sub.3, OCH.sub.2CF.sub.3, OCF.sub.2CF.sub.3, and the like.
[0053] The term alkynyl refers to a hydrocarbon with at least one
triple bond, including for example, C.sub.1 to C.sub.10 groups
including but not limited to ethynyl, 1-propynyl, 1- and 2-butynyl,
1-methyl-2-butynyl, and the like.
[0054] The term "aryl," as used herein, and unless otherwise
specified, refers to phenyl, biphenyl, or naphthyl, and preferably
phenyl. The aryl group can be optionally substituted with one or
more of the moieties selected from the group consisting of alkyl,
heteroaryl, heterocyclic, carbocycle, alkoxy, aryloxy, aryloxy;
arylalkoxy; heteroaryloxy; heteroarylaikoxy, carbohydrate, amino
acid, amino acid esters, amino acid amides, alditol, halo,
haloalkyl, hydroxyl, carboxyl, acyl, acyloxy, amino, amido,
alkylamino, dialkylamino, arylamino, nitro, cyano, thiol, imide,
sulfonic acid, sulfate, sulfonyl, sulfanyl, sulfinyl, sulfamoyl,
carboxylic ester, carboxylic acid, amide, phosphonyl, phosphinyl,
phosphoryl, thioester, thioether, oxime, hydrazine, carbamate,
phosphonic acid, phosphate, phosphonate, phosphinate, sulfonamido,
carboxamido, hydroxamic acid, sulfonylimide or any other desired
functional group that does not inhibit the pharmacological activity
of this compound, either unprotected, or protected as necessary, as
known to those skilled in the art, for example, as taught in
Greene, et al., "Protective Groups in Organic Synthesis," John
Wiley and Sons, Second Edition, 1991. Alternatively, adjacent
groups on the aryl ring may combine to form a 5 to 7 membered
carbocyclic, aryl, heteroaryl or heterocylic ring.
[0055] The term "aralkoxy" refers to an aryl group attached to an
alkyl group that is attached to the molecule through an oxygen
atom. The aryl and alkyl groups can be optionally substituted as
described above.
[0056] The term "aralkyl," as used herein, and unless otherwise
specified, refers to an aryl group as defined above linked to the
molecule through an alkyl group as defined above. The aryl and
alkyl portions can be optionally substituted as described
above.
[0057] The term "aryloxy," as used herein, refers to an aryl group
bound to the molecule through an oxygen atom. The aryl group can be
optionally substituted as set out above for aryl groups. The terms
"heteroaryl" and "heteroaromatic," as used herein, refer to
monocyclic or bicyclic aromatic ring systems of five to ten atoms
of which at least one atom is selected from O, N, and S, in which a
carbon or nitrogen atom is the point of attachment, and in which
one additional carbon atom is optionally replaced with a heteroatom
selected from O or S, and in which from 1 to 3 additional carbon
atoms are replaced by nitrogen heteroatoms.
[0058] Heteroaryl thus includes aromatic and partially aromatic
groups that contain one or more heteroatoms. Examples of this type
include but are not limited to are furan, benzofuran, thiophene,
benzothiophene, pyrrole, pyrazole, imidazole, oxazole, benzoxazole,
thiazole, benzthiazole, isothiazole, thiadiazole, triazole,
benzotriazole,furazan, benzofurazan, thiafurazan, benzothiafurazan,
tetrazole, oxadiazole, triazine, pyridine, pyridazine, pyrimidine,
pyrazine, triazine, indolizine, indole, isoindole, purine,
quinoline, benzimidazole, pteridine, isoquinoline, cinnoline,
quinazoline, and quinoxaline.
[0059] The term "heteroaralkyl," as used herein, and unless
otherwise specified, refers to a heteroaryl group as defined above
linked to the molecule through an alkyl group as defined above.
[0060] The term "heterocyclealkyl," as used herein, refers to a
heterocyclic group bound to the molecule through an alkyl group.
The heterocyclic group and the alkyl group can be optionally
substituted as described above. The term "heterocycloalkyl" can
also refer to a saturated heterocyclic moiety having from two to
six carbon atoms and one or more heteroatom from the group N, O,
and S (or oxidized versions thereof) which may be optionally
benzofused at any available position. This includes, for example,
azetidinyl, pyrrolidinyl, tetrahydrofuranyl, piperidinyl,
benzodioxolyl and the like. The term "heterocycloalkyl" also refers
to an alicyclic moiety having from three to six carbon atoms and
one or more heteroatoms from the group N, O, and S and having in
addition one double bond. Such moieties may also be referred to as
"heterocycloalkenyl" and includes, for example, dihydropyranyl, and
the like.
[0061] The term "heterocyclic" refers to a nonaromatic cyclic group
that may be partially (contains at least one double bond) or fully
saturated and wherein there is at least one heteroatom, such as
oxygen, sulfur, nitrogen, or phosphorus in the ring. The term
heteroaryl or heteroaromatic, as used herein, refers to an aromatic
that includes at least one sulfur, oxygen, nitrogen or phosphorus
in the aromatic ring. Nonlimiting examples of heterocylics and
heteroaromatics are pyrrolidinyl, tetrahydrofuryl, piperazinyl,
piperidinyl, morpholino, thiomorpholino, tetrahydropyranyl,
imidazolyl, pyrolinyl, pyrazolinyl, indolinyl, dioxolanyl,
1,4-dioxanyl, aziridinyl, furyl, furanyl, pyridyl, pyrimidinyl,
benzoxazolyl, 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl,
1,3,4-thiadiazole, indazolyl, 1,3,5-triazinyl, thienyl,
isothiazolyl, imidazolyl, tetrazolyl, pyrazinyl, benzofuranyl,
quinolyl, isoquinolyl, benzothienyl, isobenzofuryl, pyrazolyl,
indolyl, isoindolyl, benzimidazolyl, purinyl, carbazolyl, oxazolyl,
thiazolyl, benzothiazolyl, isothiazolyl, 1,2,4-thiadiazolyl,
isooxazolyl, pyrrolyl, quinazolinyl, cinnolinyl, phthalazinyl,
xanthinyl, hypoxanthinyl, pyrazole, imidazole, 1,2,3-triazole,
1,2,4-triazole, 1,2,3-oxadiazole, thiazine, pyridazine, or
pteridinyl wherein a heteroaryl or heterocyclic group can be
optionally substituted with one or more substituent selected from
the same substituents as set out above for aryl groups. Functional
oxygen and nitrogen groups on the heteroaryl group can be protected
as necessary or desired. Suitable protecting groups can include
trimethylsilyl, dimethylhexylsilyl, t-butyldimethylsilyl, and
t-butyldiphenylsilyl, trityl or substituted trityl, alkyl groups,
acyl groups such as acetyl and propionyl, methanesulfonyl, and
p-toluenelsulfonyl.
[0062] The term "heteroaryloxy," as used herein, refers to a
heteroaryl group bound to the molecule through an oxygen atom. The
heteroaryl group can be optionally substituted as set out above for
aryl groups.
[0063] The term "heterocyclearalkoxy" refers to a heterocyclic
group attached to an aryl group attached to an alkyl-O-- group. The
heterocyclic, aryl and alkyl groups can be optionally substituted
as described above.
[0064] The term "electrolyte," as used herein, refers to salts
generally and specifically to ions. An electrolyte refers to an ion
that is electrically-charged, either negative or positive. Common
electrolytes include chloride (Cl.sup.-), bromide (Br.sup.-),
bicarbonate (HCO.sub.3.sup.-), sulfate (SO.sub.4.sup.2-), sodium
(Na.sup.+), potassium (K.sup.+), calcium (Ca.sup.2+), and magnesium
(Mg.sup.2+).
Abbreviations
[0065] Abbreviations used in the examples are: DCC for
1,3-dicyclohexylcarbodiimide; DMAP for 4-dimethylamino-pyridine;
LC/MS for liquid chromatography-mass spectrometer; m-CPBA for
meta-chloroperoxybenzoic acid; and PhMe/EtOAC for toluene/ethyl
acetate.
Steroidal Quinols
[0066] In one embodiment, a quinol of Formula I is provided as
follows ##STR1## or a pharmaceutically acceptable salt or prodnig
thereof; wherein the quinol of Formula I is derived from the
following estrogen analogue (ZYC1): ##STR2## ZYC1 is an analogue of
estrogen and has been demonstrated to have estrogen-like activity.
The physicochemical properties of ZYC1 inhibit facile transcorneal
penetration upon topical administration (i.e., eye-drops). In
accordance with the present invention, ZYC1 is oxidized to produce
an steroidal quinol, 10,17-dihydroxyestra-1,4,9(11)-triene-3-one
("ZYC1-quinol"). The ZYC1-quinol, as discussed in more detail
below, has demonstrated improved physicochemical properties,
including decreased lipophilicity, to facilitate transcorneal
penetration.
[0067] In another embodiment, a quinol of Formula II is provided as
follows: ##STR3## or a pharmaceutically acceptable salt or prodrug
thereof, wherein the quinol of Formula II is derived from
3-hydroxyestra-1,3,5(10-triene-17-one (estrone).
[0068] In yet another embodiment, a quinol of Formula III is
provided as follows: ##STR4## or a pharmaceutically acceptable or
prodrug thereof, wherein the quinol of Formula III is derived from
3,17-dihydroxyestra-1,3,5(10)-triene (estradiol).
[0069] The compounds of Formulas I-III can also be functionalized
at the 3- or 17-keto group as an oxime or alkoxime. Such compounds
are useful as preliminary compounds to the quinol, for use as
pro-prodrug compounds. These compounds would be useful for a
variety of therapeutic purposes including, for example, use as a
.beta.-blocker.
[0070] The compounds and processes of the invention will be better
understood in connection with the Examples, which are intended as
an illustration of and not a limitation upon the scope of the
invention.
EXAMPLE 1
Physicochemical Properties of ZYC1
[0071] Human retinal ganglial cells (RGC) were incubated with
glutamate (5 mM), the estrogen analogue
3,17-dihydroxyestra-1,3,5(10),9(11)-tetraene (ZYC1) or combination
of glutamate and various concentrations of ZYC1. As illustrated in
FIG. 1, glutamate killed about 70% of RGC while the compound of
Formula ZYC1 alone had no effect on RGC viability. In the presence
of all three concentrations of ZYC1, glutamate killed significantly
fewer cells.
[0072] RGC were treated with glutamate (5 mM) in the presence or
absence of ZYC1. As illustrated in FIG. 2, this estrogen analogue,
ZYC1, reduced the number of RGC killed by glutamate. Where ZYC1 was
incubated in the presence of various concentrations of estrogen
receptor antagonist, IC1182,780 (ICI) (which at the lowest
concentration used, was more than 100-times its IC50), little
antagonism of ZYC1 protection of RGC was seen. This data suggests
that ZYC1 protects RGC through a non-estrogen receptor mediated
mechanism. However, the physicochemical properties of ZYC1 permit
negligible transcornneal penetration upon topical
administration.
EXAMPLE 2
Improved Physicochemical Properties of Steroidal Quinols
[0073] To test the hypothesis that directed modification of an
estrogen improves physicochemical properties of transcorneal
penetration, estrone was used as a lead compound. The following
Table I indicates a very significant drop in lipophilicity of
Formula I, Formula II, and Formula III, compared to the parent
phenolic A-ring steroids, ZYC1, estrone, and estradiol. The log of
the n-octanol/water partitioning coefficient (log P or log
D.sub.7.4) is the measure of attraction to lipid phase versus an
aqueous phase. Log P is a crucial factor governing passive membrane
partitioning, influencing permeability opposite to its effect on
solubility (i.e., increasing log P enhances permeability while
reducing water solubility). Thus, the results of Table I
demonstrate that the lipophilic-hydrophilic balance of Formula I,
Formula II, and Formula III are closer to the ideal value for
facile transcorneal penetration and favorable bioavailability than
the parent phenolic A-ring steroids, ZYC1, ZYC3, estrone, and
estradiol. It has been demonstrated that the ideal log P value for
the brain is approximately 2. Though an ideal log P value for the
cornea has not yet been determined, a log P value of two should be
a reasonable value for the cornea. TABLE-US-00001 TABLE I COMPOUND
Log P P 3-hydroxyestra-1,3,5(10)-triene-17-one (estrone) 4.54
64,670 3,17.beta.-dihydroxyestra-1,3,5(10)-triene (estradiol) 4.01
10,230 3,17-dihydroxyestra-1,3,5(10),9(11)-tetraene (ZYC1) 3.57
3,715 2-(1-adamantyl)-3-hydroxyestra-1,3,5(10)-trien-17-one [2-(1-
6.83 6.76 10.sup.6 adamantyl)estrone] (ZYC3)
10.beta.-hydroxyestra-1,4-diene-3,17-dione ("estrone quinol") 2.20
158 10.beta.,17.beta.-dihydroxyestra-1,4-diene-3-one ("estradiol
quinol") 1.67 47 10.beta.,17-dihydroxyestra-1,4,9(11)-triene-3-one
("Formula * 1.30 20 quinol")
2-(1-adamantyl)-10.beta.-hydroxyestra-1,4-diene-3,17-dione ["2-(1-
4.30 19,953 adamantyl)estrone quinol"] (
The log P values pertain to n-octanol/water partitioning were
predicted by the method incorporated into CAChe WorkSystem Pro 5.0
(Fujitsu America, Inc., Beaverton, Oreg.).
EXAMPLE 3
General Methods for Preparing a Steroidal Quinol
[0074] By way of example, Formula II (estrone quinol;
10.beta.-hydroxyestra-1,4-diene-3,17-dione) was prepared by the
following Scheme I: ##STR5##
[0075] As understood by the skilled artisan, steroidal quinols
according to the present invention may be synthesized using a
"one-pot" phenol to quinol transformation. The synthesis method
utilizes m-CPBA as an oxidant, dibenzoyl peroxide
[(PheCO).sub.2O.sub.2] as a radical initiator and visible-light
irradiation that, in refluxing aprotic solvent, produces excellent
yields of the quinols of the present invention.
[0076] By way of example, Solaja et al., Tetrahedron Letters,
37:21, 3765-3768 (1996) discloses a "one-pot" method for
synthesizing estrone-quinol. Oxidation of estrone to synthesize
10.beta.-hydroxyestra-1,4-diene-3,17-dione is performed by heating
a stirred solution of estrone (10.00 g, 37.0 mmol), m-CPBA (22.53
g, 111.0 mmol; 85% Jansen Chimica), and (PheCO).sub.2O.sub.2 (900
mg, 3.70 mmol) in 2 L mixture of CCl.sub.4/Me.sub.2CO (4/1) to
reflux for 3 hours while irradiated with a 60 Watt tungsten lamp.
Upon evaporation of the solvent, extraction is performed with
CHCl.sub.3 (3.times.200 mL), washing with NaHCO.sub.3 (2.times.100
mL) and H.sub.2O (100 mL), and drying over anhydrous
Na.sub.2SO.sub.4. The residue is then chromatographed on SiO.sub.2
column. Elution may be performed with PhMe/EtOAc (1/1 and 7/3,
respectively) and crystallization from benzene produces 5.19 g
(49%) of estrone quinol as colorless needles.
[0077] Data regarding the resulting estrone quinols, as observed by
Solaja et al. are as follows: mp=219-221.degree. C. (benzene);
.sup.1H-NMR (250 MHz, DMSO-d.sub.6): 7.13 (d, j=10.4 Hz, H--C(1)),
6.07 (dd, J=10.4, 2.4 Hz, H--C(2)), 5.92 (irreg. T, J.sub.4,2=2.4,
J.sub.4,6.beta.=1.2 Hz, H--C(4)), 5,67 (s, H-o, exchangeable with
D.sub.2O), 2.67 (tdd, J=15.2, 6.4, 1.2 Hz, H.sub..beta.--C(6)),
1.97-1.83 (m, H.sub..beta.--C(8) and H.sub..beta.--C(11), 1.30-1.18
(m, H.sub..alpha.--C(11)), 0.97 (s, H.sub.3C--C(13)); .sup.13C NMR
(62.9 MHz, DMSO-d.sub.6): 220.33 (C(17)), 185,53 (C(3)), 165.09
(C(5)), 150.25 (C(1)), 128.30 (C(2)), 123.09 (C(4)), 70.10 (C(10)),
51.18 (C( ), 50.10 (C(14)), 47.75 (C(13)), 35.62 (C(16)), 34.58
(C(8)), 32.19 (C(7)), 31.80 (C(6)), 31.03 (C(11)), 22.00 (C(12)),
21.90 (C(15)), 13.73 (C(18)); MS (EI, m/z): 286(M.sup.+, 84),
268(M.sup.+-H.sub.2O, 39), 150(68), 145(100), 124(75), 107(50),
91(50), 79(54), and 55(60).
[0078] Alternatively, estrome quinols of the present invention can
be prepared using
2-(1-adamantyl)-3-hydroxyestra-1,3,5(10)-trien-17-one
[2-(1-adamantyl)estrone], which can be made using methods
previously described by Lunn, W. H. and E. Farkas, "The adamantly
carbonium ion as a dehydrogenating agent, its reactions with
estrone," Tetrahedron, 24:6773-6776 (1968). Estrone (270 mg, Immol)
and 1-adamantanol (170 mg, 1 mmol) were added to anhydrous
n-pentane (6 mL) and the stirred mixture was cooled with an ice
bath. Boron trifluoride etherate (BF.sub.3 EtOEt, 0.4 mL) was added
over a 10 minutes period. After an additional 15 min, the ice bath
was removed and stirring was continued for an additional 45 min at
room temperature. During the 45 min, the solid present in the
reaction mixture was dissolved and yellow oil formed. Crushed ice
was then added while shaking and swirling the reaction flask and
pink solid was formed. The filtered crude pink product was washed
with water until the filtrate had a neutral pH and the solid was
dried in a vacuum oven at 50.degree. C. The pink crude powder (0.4
g) was purified by flash chromatography (silica gel, eluted with
20% ethyl acetate in hexanes to yield the pure product; 0.31 g,
76.7%). The product was recrystallized from a mixture of chlorofonn
and isopropyl alcohol and had: mp 322-324.degree. C., lit mp
295-296.degree. C.; .sup.1H NMR (CDCl.sub.3, 300 MHz) .delta. 0.91
(s, 3H, C.sub.18--CH.sub.3), 2.8 (m, 2H, C.sub.6--CH.sub.2), 4.71
(s, 1H, C.sub.3--OH), 6.42 (s, 1H, Aromatic H), 7.15 (s, 1H,
Aromatic H).
[0079] 2-(1-Adamantyl)estrone (also referred to herein as ZYC3) was
oxidized with lead-acetate to the corresponding quinol acetate
using the following procedures. To a solution of 3 g
2-(1-adamantyl)estrone in 50 ml of glacial acetic acid 11 g of
lead(IV)-acetate was added. The solution was stirred at room
temperature for 1 day. Then, the solution was concentrated in vacuo
to an oil that was treated with 50 ml of water and 50 ml of
chloroform. The organic layer was separated and washed with 10%
NaHCO.sub.3 and water. After drying over Na.sub.2SO.sub.4 the
chloroform was removed and the residue was purified by column
chromatography on silica gel using hexane ethyl acetate 4:1 (v/v)
eluent. The pure quinol acetate (as illustrated in FIG. 3), which
is also a potential prodrug, has R.sub.f=0.5 on silicagel TLC with
the same eluent. Typical resonances (ppm) in .sup.1H-NMR
(CDCl.sub.3) spectrum indicating the conversion to 2-substituted
estrone quinol acetate were observed: 6.4 (s, 1H, H-1); 6.0 (s, 1H,
H-4); 2.0 (s, 3H, 10-acetyl).
[0080] The quinol acetate (was then hydrolyzed to the quinol (where
R.dbd.H; prepared as described above) in methanol using a slight
excess of NaOMe in methanol (25% w/v) overnight at room
temperature. Then the solution was concentrated and glacial acetic
acid was added to adjust the pH slightly acidic. Upon adding water
the quinol precipitated out as a pale yellow solid that was again
purified by column chromatography the same way as its acetate
(R.sub.f=0.53). Typical resonances (ppm) in .sup.1H-NMR
(CDCl.sub.3) indicating the conversion to 2-substituted estrone
quinol were observed: 6.6 (s, 1H, H-1); 5.9 (s, 1H, H-4). MS (EI):
m/z 420 (M.sup.+*).
[0081] To prepare (alk)oxime estradiol quinols of the subject
invention, such as those illustrated in FIG. 4, 0.5 g of
hydroxylamine hydrochloride is added to 0.5 g of estradiol quinol
or alkoxyamine hydrochloride) in 5 ml of ethanol, 0.5 ml of
pyridine was added and the solution was refluxed overnight. After
cooling, the ethanol was removed and ice-cold water was added. The
mixture was stirred until the oxime crystallized.
EXAMPLE 4
Physicochemical Properties of ZYC3
[0082] RGC were incubated with glutamate (5 mM), with
2-(1-adamantyl)-3-hydroxyestra-1,3,5 (10)-trien-17-one (ZYC3), or
with a combination of glutamate and various concentrations of ZYC3.
As illustrated in FIG. 5, glutamate killed about 70% of RGC while
the compound of ZYC3 alone has no affect on RGC viability. In the
presence of three different concentrations of ZYC3, glutamate
killed significantly fewer cells (No statistically significant
difference from RGC survival without exposure to glutamate).
EXAMPLE 5
Prodrug Activity
[0083] By way of example, conversion of Formula II by NAD(P)H as an
endogenous reducing agent was tested. Estrone quinol (0.1 mM) and
1.0 mM of NADPH or NADH in 0.1M sodium phosphate buffer (1 ml final
volume, pH 7.5) was incubated at 37.degree. C. At incremental time
points, 100 .mu.l aliquots were removed into ice-cold centrifuge
tubes, and 100 .mu.l of glacial acetic acid was added. After
immediate extraction with ethyl acetate, the organic layer was
evaporated under nitrogen stream. Reconstitution of the samples
with the liquid chromatography mobile phase was followed by LC/MS
analyses, the results of which are illustrated in FIGS. 6-8. For
the control experiment, no reducing agent was used.
[0084] Liquid chromatography separation was done using a Supelco
(Bellfonte, Pa.) 5 cm.times.2.1 mm i.d. Discovery HS C-18
reversed-phase column with 0.25 ml/min
water:methanol:2-propanol:acetic acid:dichloromethane (53:35:5:5:2,
v/v) as a mobile phase. The sample residues were dissolved in 40
.mu.l of mobile phase, respectively, and 5 .mu.l of the solution
was injected for analysis. Mass spectra were recorded on a
quadruple ion-trap instrument (LCQ.RTM., ThermoFinnigan, San Jose,
Calif.) using positive-ion atmospheric-pressure chemical ionization
(APCI) as the method of ionization. MS/MS and MS.sup.3 product-ion
scans were obtained after collision-induced dissociation (CID) with
helium as the target gas. Comparison with authentic reference
compound (retention time, t.sub.R, and mass spectra) was used for
unambiguous identification of estrone. As an internal standard,
1,3,5(10)-estratrien-17.alpha.-ethynyl-17.beta.-ol was added before
each sample extraction. Estrone and estrone quinol levels were
detennined by LC/APCI-MS/MS and calibration with solutions of known
concentrations of estrone (0.02 .mu.M to 11 .mu.M) and estrone
quinol (0.2 .mu.M to 125 .mu.M) extracted for analyses. The
chromatographic peak areas for estrone and estrone quinol were
obtained from m/z 271.fwdarw.253 and m/z 287.fwdarw.269 MS/MS
transitions, respectively. Formation of estrone was clearly
detectable even after a short period of time, when the incubation
was carried out in the presence of NADH and, especially, NADPH.
[0085] The rate of conversion at 37.degree. C. and with a 10-fold
access of the ubiquitous reducing agent NADPH is
6.0.times.10.sup.-7.+-.4.times.10.sup.-8 Mmin.sup.-1, which
indicates a rapid process required for the proposed action of a
quinol as a prodrug. Enzymes may also catalyze reductions in the
eye. See Sichi H and D. W. Nebert, "In: Extrahepatic Metabolism of
Drugs and Other Foreign Compounds (Gram T E, Ed.)," S. P. Medical
and Scientific Books, New York, pp. 333-363 (1980), and Starka L
and J. Obenberger. (In vitro estrone-estradiol-17beta
interconversion in cornea, lens, iris and retina of rabbit eye,"
Arch Klin Exp Ophthalmol, 196:199-204 (1975).
EXAMPLE 6
General Methods for Preparing Prodrugs
[0086] In general, where a steroidal quinol according to the
subject invention contains a hydroxyl group (i.e., 17-OH group or
10.beta.-OH group), an "ester" moiety can replace the hydroxyl
portion to form a non-acidic (neutral) ester compound. The addition
of a polar functional group (i.e., tertiary amide or phosphate
ester) enhances the phenolic A-ring steroid-derived quinol's
affinity to water and thus facilitates the transport of the quinol
through the lipid-poor soma in the cornea. The following compounds
of Formula III, Formula IIIa and Formula IIIb, illustrate polar
functional groups attached at the 17-OH group. ##STR6##
[0087] wherein
[0088] each R and R' is independently hydrogen, alkyl, alkenyl,
alkynyl, alkoxy, aryl, aralkoxy, aralkyl, aryloxy, hydroxyalkyl,
alkoxyalkyl, heteroaralkyl, heterocyclealkyl, heteroaryloxy; and
heterocyclearalkoxy;
[0089] X is an electrolyte; and
[0090] n is an integer from 1 to 20.
[0091] By way of example, a prodrug of Formula I (n=1, R.dbd.H) may
be obtained by converting Formula III into an ester compound as
illustrated in the following Scheme IIa. To a solution of
10.beta.,17.beta.-dihydroxyestra-1,4-diene-3one (Formula I,
estradiol quinol) in chloroform or ethyl acetate bromoacetic
anhydride, DCC, and DMAP are added. The resulting mixture is
stirred at 20-25.degree. C. for 48 hours. The organic solution is
extracted with water then dried over Na2SO4 and evaporated. The
residue is purified by chromatography (silica gel: Aldrich, Merck
grade 60, 230-400 mesh, 32.times.2 cm; elution with hexane
containing gradually increasing concentrations of ethyl acetate
from 0 to 6%). The purified residue in hexane is then placed in a
closed system under argon, and trimethylamine (gas) was added at
20-25.degree. C. then the precipitate was filtered, and rinsed with
hexane. The resultant prodrug of estradiol quinol
(10.beta.,17.beta.-dihydroxyestra-1,4-diene-3-one-17-acetyl-trimethylammo-
nium bromide) should have adequate solubility and sufficient
stability to allow for formulation and storage. Further, the
exemplary prodrug of estrone quinol is easily converted through an
enzymatic or chemical process to the active compound, estrone,
within the body, preferably the eye. In the following Scheme IIa, a
prodrug of Formula I can be obtained. ##STR7##
[0092] wherein R, R', X, and n are as defined above.
[0093] Phosphate esters can also be attached as a polar functional
group to enhance water affinity of steroidal quinols. For example,
phosphate ester prodrugs of estrogens according to the present
invention can be prepared by an ester linkage to one of the
hydroxyl groups of the head group of an steroidal quinol.
[0094] By way of example, the prodrug of estradiol, in accordance
with the present invention, may be prepared using general methods
as depicted in the following Schemes IIIa and IIIb. ##STR8##
##STR9##
EXAMPLE 7
Physicochemical Properties of
2-(1-adamantyl)-estra-1,3,5(10)-triene-3,17.beta.-diol as compared
with 2-(1-adamantyl)-3-hydroxyestra-1,3,5(10)-trien-17-one
(ZYC3)
[0095] Because lipid peroxidation (LPO) is a common marker of
damage induced by reactive oxygen species (ROS) (Gutteridge JMC,
"Lipid-peroxidation and antioxidants as biomarkers of
tissue-damage," Clin Chem, 41: 1819-1828 (1995); Kaur and Geetha,
"Screening methods for antioxidants--A review," Mini-Rev Med Chem,
6: 305-312 (2006)), two assays were employed to measure capacity to
inhibit LPO: the ferric thiocyanate (FTC) and the thiobarbituric
acid reactive substances (TBARS) methods. With these assays, the
autoxidation of linoleic acid (a lipid model) is measured in the
absence and, then, presence of different concentration of
antioxidants. The FTC method measures the amount of peroxide in
initial stages of lipid oxidation (Kikuzaki H and Nakatani N.,
"Antioxidant effects of some ginger constituents," J Food Sci, 58:
1407-1410 (1993)). During the oxidation process, peroxide is
gradually decomposed to lower molecular-weight compounds such as
malondialdehyde (MDA) that is measured by the TBARS method
(Kikuzaki and Nakatani, J Food Sci, 58: 1407-1410 (1993)).
Therefore, the FTC and TBARS assays are complementary, when they
are used to evaluate antioxidants for their capacity to inhibit
LPO.
[0096] The FTC assay is based on the oxidation of ferrous to ferric
ion by the lipid hydroperoxides (LOOH), followed by a subsequent
complexation of Fe.sup.3+ with the thiocyanate anion (Mihaljevic B
A, Katusin-Razem B and Razem D, "The reevaluation of the ferric
thiocyanate assay for lipid hydroperoxides with special
considerations of the mechanistic aspects of the response." Free
Rad Biol Med, 21: 53-63 (1996)). The amount of lipid hydroperoxides
is measured spectrophotometrically as ferric thiocyanate complex,
which gives a strong absorbance at 500 nm.
[0097] The TBARS assay is a widely adopted and sensitive method for
measurement of lipid peroxidation (Callaway J K, Beart P M and
Jarrott B. "A reliable procedure for comparison of antioxidants in
rat brain homogenates." J Pharmacol Toxicol Meth, 39: 155-162
(1998)). The oxidation of unsaturated fatty acids leads to the
formation of MDA as a breakdown product (Mihaljevic et al., Free
Rad Biol Med, 21: 53-63 (1996)). The reaction of MDA with
thiobarbituric acid (TBA) produces a pink chromogen when heated at
low pH with a typical maximum absorbance at 532 nm (Esterbauer H
and Cheeseman K H. "Determination of aldehydic lipid peroxidation
products: malondialdehyde and 4-hydroxynonenal." Methods Enzymol,
186: 407-421 (1990)). The MDA-TBA complex measured by the TBARs
assay is a gauge of LOOH formation (Janero D R. "Malondialdehyde
and thiobarbituric acid-reactivity as diagnostic indices of lipid
peroxidation and peroxidative tissue injury." Free Rad Biol Med, 9:
515-540 (1990)). Inhibitions were calculated from absorbances
measured in the presence of the compounds at different
concentrations and absorbance of the control reaction (no
antioxidant added), and IC.sub.50 values (concentration that
inhibits 50% of lipid peroxidation) were determined by sigmoidal
fitting (Prizm 3.0, GraphPad) of the inhibition versus
concentration curves. A smaller IC.sub.50 value represents a higher
potency to inhibit LPO. Both the FTC and TBARS methods were
utilized to assess the differences between
2-(1-adamantyl)-estra-1,3,5(10)-triene-3,17.beta.-diol and
2-(1-adamantyl)-3-hydroxyestra-1,3,5(10)-trien-17-one (ZYC3). The
results, as illustrated in Table 2 below, indicate that
2-(1-adamantyl)-estra-1,3,5(10)-triene-3,17.beta.-diol has higher
potency than ZYC3 to inhibit lipid peroxidation. TABLE-US-00002
TABLE 2 IC.sub.50: FTC IC.sub.50: TBARS Compound method (.mu.M)
method (.mu.M) 2-(1-adamantyl)-3- 5.3 .+-. 1.2 5.8 .+-. 1.3
hydroxyestra-1,3,5(10)-trien-17-one 2-(1-adamantyl)- 1.5 .+-. 0.1
0.75 .+-. 0.11 estra-1,3,5(10)-triene-3,17.beta.-diol
EXAMPLE 8
2-(1-adamantyl)-10.beta.,17.beta.-dihydroxyestra-1,4-dien-3-one
Prodrug Activity
[0098] As illustrated below in Scheme IV,
2-(1-Adamantyl)-.DELTA..sup.1-dehydro-19-nortestosterone (also
referred to herein as
2-(1-adamantyl)-10.beta.,17.beta.-dihydroxyestra-1,4-dien-3-one) is
a prodrug of
2-(1-adamantyl)-estra-1,3,5(10)-triene-3,17.beta.-diol.
##STR10##
[0099] Experiments conducted on
2-(1-adamantyl)-.DELTA..sup.1-dehydro-19-nortestosterone indicate
that it is highly suitable for pharmaceutical purposes (beneficial
properties exhibited such as permeability across biological
membranes if released from non-erodible drug delivery systems such
as ocular inserts or polymeric nanoparticles, and the like). As
indicated in Table 3 below, the lipophilicity property of the
2-(1-adamantyl)-.DELTA..sup.1-dehydro-19-nortestosterone prodrug is
preferred to that of the converted, active compound:
2-(1-adamantyl)-estra-1,3,5(10)-triene-3,17.beta.-diol:
TABLE-US-00003 TABLE 3 Compound logP.sub.calc.sup.a, (i)
logP.sub.exp.sup.a, (ii) 2-(1-adamantyl)-estra- 6.40 6.51
1,3,5(10)-triene-3,17.beta.-diol 2-(1-adamantyl)-.DELTA..sup.1-
3.81 3.26 dehydro-19-nortestosterone .sup.aP denotes the
n-octanol/water partitioning coefficient, which is a measure of
attraction to lipid phase versus an aqueous phase. The logarithm of
n-octanol/water partitioning coefficients (logP) was (i) calculated
from molecular model by the method incorporated # into the program
BioMedCAChe (version 6.1, Fujitsu America, Inc., Beaverton, OR) and
(ii) measured experimentally by the shake-flask method (Leo A,
Hansch C, and Elkins D. "Partition coefficients and their uses."
Chem. Rev, 71: 525-616. (1971)).
EXAMPLE 9
General Methods for Preparing
2-(1-adamantyl)-.DELTA..sup.1-dehydro-19-nortestosterone
Prodrug
[0100] Scheme V below illustrates a method for synthesizing
2-(1-adamantyl)-.DELTA..sup.1-dehydro-19-nortestosterone:
##STR11##
[0101] The synthesis method of Scheme V is based on
microwave-assisted oxidation of the corresponding phenolic
compounds with lead (IV) acetate. The corresponding phenolic
compound is prepared according to Lunn & Farkas (Tetrahedron
24, 6773-6776, 1968). Briefly, 17.beta.-estradiol (1 mmol) and
1-adamantanol (1.05 mmol) was added to 20 ml dry hexane and
followed by the drop wise addition of 0.5 ml of BF.sub.3.Et.sub.2O
under ice cooling. The cooling was, then, removed and the stirring
continued overnight. The reaction mixture was poured onto crashed
ice and the obtained precipitate was filtered off, washed with
water and dried. Column chromatographic purification was done on
silicagel, using hexane: ethyl acetate 4:1 (v/v) eluent. (White
solid: m.p. 180-182.degree. C.) APCI-MS: (M+H).sup.+ m/z 407.
Conversion of the phenolic compound to
2-(1-adamantyl)-.DELTA..sup.1-dehydro-10.beta.-hydroxy-19-nortestosterone
was done by microwave-accelerated oxidation with lead(IV) acetate.
The phenolic compound (1 mmol) was dissolved in 6 mol glacial
acetic acid and lead (IV) acetate (1.7 mmol) was added. The
closed-vessel reaction under pressure control was performed in a
glass vessel (capacity 10 mL) sealed with a septum. A CEM
(Matthews, N.C.) Discover monomode microwave apparatus, operating
at a frequency of 2.45 GHz with continuous irradiation power from 0
to 300 W was used. The temperature was measured by infrared
detection with continuous feedback temperature control, and
maintained at a constant value by power modulation. The reaction
temperature was set at 45.degree. C. After irradiation for 25 min,
the reaction vessel was cooled rapidly to ambient temperature by
compressed air. With a nitrogen stream, the solution was
concentrated and enough NaOMe in MeOH (25% w/v) was added to
increase the pH to around 9. Irradiation of the solution for
another 5 minutes at 45.degree. C. produced
2-(1-adamantyl)-.DELTA..sup.1-
dehydro-10.beta.-hydroxy-19-nortestosterone that was isolated after
concentrating the solution, adjusting the pH with acetic acid to
slightly acidic and treatment with ice-cold water. The crude
product was purified by column chromatography on silica gel using
hexane:ethyl acetate 3:2 (v/v) eluent. Yield 45%. APCI-MS:
(M+H).sup.+ m/z 423. Also see Example 11 below.
EXAMPLE 10
Biological Activity of
2-(1-adamantyl)-.DELTA..sup.1-dehydro-19-nortestosterone
Prodrug
[0102] As indicated in FIG. 9, the prodrug
2-(1-adamantyl)-.DELTA..sup.1-dehydro-19-nortestosterone appears to
be equally effective in treating retinitis pigmentosa as with the
active agent
2-(1-adamantyl)-estra-1,3,5(10)-triene-3,17.beta.-diol.
Heterozygous S334ter rhodopsin mutation transgenic rats
(Sprague-Dawley parental) were used as a model for retinitis
pigmentosa. 10 mM of
2-(1-adamantyl)-.DELTA..sup.1-dehydro-19-nortestosterone and 10 mM
of 2-(1-adamantyl)-estra-1,3,5(10)-triene-3,17.beta.-diol were
introduced to the heterozygous S334ter rhodopsin mutation
transgenic rats via intravitreal injection. 10 mM of DMSO (vehicle)
was injected as a control. The prodrug
2-(1-adamantyl)-.DELTA..sup.1-dehydro-19-nortestosterone and the
active agent 2-(1-adamantyl)-estra-1,3,5(10)-triene-3,17.beta.-diol
demonstrated equivalent protection of the outer nuclear layer (ONL)
of the retina in the heterozygous S334ter rhodopsin mutation
transgenic rats. Specifically, 3-5 ONL layers were protected after
treatment with
2-(1-adamantyl)-.DELTA..sup.1-dehydro-19-nortestosterone and
2-(1-adamantyl)-estra-1,3,5(10)-triene-3,17.beta.-diol, whereas
only 1-2 ONL layers were protected without treatment (vehicle
control with DMSO).
EXAMPLE 11
Microwave-Assisted Synthesis of p-Quinols by Lead(IV) Acetate
Oxidation
[0103] One conventional method for the synthesis of steroidal
p-quinols uses lead (IV) acetate oxidizing agent and very long
(30.sup.+ h) reaction time resulting in numerous side-reactions
that make the isolation of the desired p-quinol cumbersome and very
inefficient (Gold A M, Schwenk E., "Synthesis and reactions of
steroidal quinols." J Am. Chem. Soc., 80:5683 (1958)). In another
method, m-chloroperoxybenzoic acid is used for the oxidation with
dibenzoil peroxide radical initiation upon light irradiation, and
3.5-24 h of reaction time is required to complete the reaction
depending on the phenolic compound to be oxidized ((a) Solaja, B.
A.; Milic, D. R.; Gasic, M. J. Tetrahed. Lett., 37, 3765 (1996);
(b) Milic, D. R.; Gasic, M. J.; Muster, W.; Csanadi, J. J.; Solaja,
B. A. Tetrahedron, 53, 14073 (1997)). While certain p-quinols
(2a,b; see Table 4 below) were able to be obtained with about 50%
yield from estrone (1a; see Table 4 below) and 17.beta.-estradiol
(1b; see Table 4 below) within 6 h by using the latter method,
A-ring substituted estrogens (e.g., 1c; see Table 4 below),
17.beta.-alkyl ether derivatives of 1b (e.g., 1d; see Table 4
below) or simple p-alkylphenols such as 5a,b (see Table 4 below)
did not convert to the corresponding p-quinols with appreciable
yields, neither by radical-initiated oxidation with
m-chloroperoxybenzoic acid nor with the conventional Pb(OAc).sub.4
oxidation, even after prolonged reaction times (>24 h).
[0104] Microwave-assisted organic synthesis (MAOS) has received
considerable attention in recent years because of the rapid
synthesis of a variety of organic compounds (Kappe, C. O., Angew.
Chem. Int. Eng. Ed., 43, 6250 (2005)). MAOS was used for the
preparation of p-quinols of the invention. It was reasoned that by
significantly shortening the reaction time upon microwave
irradiation when Pb(OAc).sub.4 is used for the oxidation, the
extent of side-reactions would be reduced and, thus, increase the
yield and simplify the isolation process. Further, amount of lead
salt traditionally used [1:3 molar ratio of the starting material
and Pb(OAc).sub.4] could be reduced.
[0105] Experiments were carried out in CEM (Matthews, N.C.)
Discover monomode microwave apparatus, operating at a frequency of
2.45 GHz with continuous irradiation power of 0 to 300 W, was used.
The temperature (measured by infrared detection) was maintained at
40.degree. C. by continuous feedback control and power modulation.
The closed-vessel reaction under controlled pressure was performed
in a glass vessel (capacity 10 mL) sealed with a septum. After
irradiation, the reaction vessel was cooled rapidly to ambient
temperature by compressed air cooling; the phenolic compounds (1
eq) were dissolved in glacial acetic acid, and Pb(OAc).sub.4 (1.5
eq) was added. After 15-20 minutes of irradiation, the reactions
were complete (monitored by TLC). With a nitrogen stream, the
solution was concentrated and NaOMe in MeOH (25%, w/v) was added to
increase the pH to approximately 9. Irradiation of the solution for
another 5 min produced the target p-quinols that were isolated
after treatment with ice-cold water. The crude products were
obtained (in contrast with black oils yielded by the conventional
method; Gold A M, Schwenk E., J. Am. Chem. Soc., 80:5683 (1958)) as
pale-yellow solids that were purified by flash column
chromatography on silica gel.
[0106] Indeed, as illustrated in Scheme VI below, the p-quinol
formation was complete within 15-20 min (monitored by TLC) when
microwave irradiation was applied for the oxidation of all of the
phenolic compounds of interest (1a-d, 4, and 5a,b; see Table 4
below) using only 1.5 eq of Pb(OAc).sub.4 in glacial acetic acid.
The intermediate p-quinol acetates were not isolated, but
hydrolyzed after removal of the solvent and by subsequent addition
of NaOMe in MeOH to the target p-quinols under microwave
irradiation within 5 min, while the original procedure (Gold A M,
Schwenk E., J. Am. Chem. Soc., 80:5683 (1958)) called for
approximately 12 h of reaction time to hydrolyze the intermediates.
Moreover, microwave-assisted synthesis was suitable for the rapid
oxidation of A-ring substituted estrogens (e.g.; 1c), while
conventional methods were not applicable and/or efficient for these
type of compounds. After flash chromatographic purification, 2a-d,
4, and 6a,b were obtained with consistent 40-50% yields (Table 4
below).
[0107] Taken together, the MAOS approach presented here
significantly reduced the reaction time and the amount of
Pb(OAc).sub.4 used for the oxidation of p-alkyl phenols. The
procedure provided, therefore, an increased throughput and more
ecofriendly route to obtain p-quinols of the invention. This method
also provides a convenient, fast, reliable and universally
applicable route for the synthesis of the subject quinol compounds,
and is useful for obtaining valuable intermediates that allow for
the synthesis of many complex organic molecules. TABLE-US-00004
TABLE 4 Comparison of yields and reaction times between
conventional methods and the microwave-assisted procedure for the
synthesis of p-quinols using Pb(OAc).sub.4 [scale: 2 mmol, equiv.
ratio of starting material/ Pb(OAc).sub.4 = 1:1.5]. Target
Conventional Methods Microwave-Assisted Synthesis Compound Yield
(%).sup.a Time (h) Yield (%).sup.a Time (min).sup.b 2a
20.sup.b/54.sup.c 36.sup.d/4.sup.c 50 30 2b 10.sup.b/45.sup.c
36.sup.d/6.sup.c 45 30 2c <5.sup.b,c 24.sup.c,d 40 30 2d
<5.sup.b,c 24.sup.c,d 50 25 4 <10.sup.b,c 5.sup.d 47 25 6a
<10.sup.c 24.sup.c 40 25 6b <5.sup.b,c 24.sup.c,d 45 30
.sup.aYield after purification by flash chromatography; .sup.bBy
radical-initiated oxidation with m-chloroperoxybenzoic acid,
according to Gold AM, Schwenk E., J. Am. Chem. Soc., 80: 5683
(1958); .sup.cBy Pb(OAc).sub.4 oxidation, according to reference 7
Solaja, B. A.; Milic, D. R.; Gasic, M. J. Tetrahed. Lett., 37, 3765
(1996); and Milic, D. R.; Gasic, M. J.; Muster, W.; Csanadi, J. J.;
Solaja, B. A. Tetrahedron, 53, 14073 (1997); .sup.dCombined
reaction time (oxidation and hydrolysis).
[0108] ##STR12##
[0109] All patents, patent applications, provisional applications,
and publications referred to or cited herein are incorporated by
reference in their entirety, including all figures and tables, to
the extent they are not inconsistent with the explicit teachings of
this specification.
[0110] It should be understood that the examples and embodiments
described herein are for illustrative purposes only and that
various modifications or changes in light thereof will be suggested
to persons skilled in the art and are to be included within the
spirit and purview of this application.
* * * * *