U.S. patent application number 11/845906 was filed with the patent office on 2008-02-07 for hydroxy-biphenyl-carbaldehyde oxime derivatives and their use as estrogenic agents.
This patent application is currently assigned to Wyeth. Invention is credited to Richard Eric Mewshaw, Cuijian Yang.
Application Number | 20080033049 11/845906 |
Document ID | / |
Family ID | 33436730 |
Filed Date | 2008-02-07 |
United States Patent
Application |
20080033049 |
Kind Code |
A1 |
Mewshaw; Richard Eric ; et
al. |
February 7, 2008 |
Hydroxy-Biphenyl-Carbaldehyde Oxime Derivatives and Their Use As
Estrogenic Agents
Abstract
This invention provides estrogen receptor modulators having the
structure: ##STR1## wherein R.sup.1 to R.sup.6 and R.sup.8 are as
defined in the specification; or a pharmaceutically acceptable salt
thereof.
Inventors: |
Mewshaw; Richard Eric; (King
of Prussia, PA) ; Yang; Cuijian; (Collegeville,
PA) |
Correspondence
Address: |
WOODCOCK WASHBURN LLP/WYETH
CIRA CENTRE, 12TH FLOOR
2929 ARCH STREET
PHILADELPHIA
PA
19104-2891
US
|
Assignee: |
Wyeth
Madison
NJ
|
Family ID: |
33436730 |
Appl. No.: |
11/845906 |
Filed: |
August 28, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10835228 |
Apr 29, 2004 |
7279600 |
|
|
11845906 |
Aug 28, 2007 |
|
|
|
60467394 |
May 2, 2003 |
|
|
|
Current U.S.
Class: |
514/640 ;
564/265 |
Current CPC
Class: |
A61P 13/08 20180101;
A61P 37/06 20180101; A61P 7/02 20180101; A61P 17/14 20180101; A61P
37/02 20180101; A61P 17/00 20180101; A61P 31/12 20180101; A61P
13/12 20180101; A61P 25/00 20180101; C07C 251/48 20130101; A61P
3/14 20180101; A61P 5/00 20180101; A61P 25/28 20180101; A61P 39/06
20180101; A61P 13/02 20180101; A61P 15/18 20180101; A61P 17/10
20180101; A61P 5/30 20180101; A61P 3/10 20180101; A61P 9/00
20180101; A61P 15/08 20180101; A61P 13/10 20180101; A61P 15/00
20180101; A61P 3/06 20180101; A61P 31/06 20180101; A61P 35/00
20180101; A61P 35/02 20180101; A61P 43/00 20180101; A61P 1/16
20180101; A61P 19/02 20180101; A61P 19/00 20180101; A61P 19/10
20180101; A61P 15/16 20180101; A61P 9/08 20180101; A61P 11/00
20180101; A61P 17/06 20180101; A61P 1/04 20180101; A61P 15/02
20180101; A61P 9/10 20180101 |
Class at
Publication: |
514/640 ;
564/265 |
International
Class: |
A61K 31/15 20060101
A61K031/15; A61P 19/00 20060101 A61P019/00; C07C 291/02 20060101
C07C291/02 |
Claims
1. A compound of the formula ##STR23## wherein: R.sup.1 is OH or
lower alkoxy; and R.sup.5, R.sup.6, and R.sup.7 are each,
independently, H, OH, halogen, CN, phenyl, lower alkyl, lower
alkoxy, said phenyl, lower alkyl, and lower alkoxy being optionally
substituted; or a pharmaceutically acceptable salt thereof or a
prodrug thereof.
2. The compound of claim 1 wherein R.sup.5, R.sup.6, and R.sup.7
are each, independently, H, Me, Cl, F, or methoxy.
3. The compound of claim 1 where the compound is
4'-Hydroxy-3-methoxy-1,1'-biphenyl-4-carbaldehyde oxime.
4. A pharmaceutical composition comprising: ##STR24## wherein:
R.sup.1 is OH or lower alkoxy; and R.sup.5, R.sup.6, and R.sup.7
are each, independently, H, OH, halogen, CN, phenyl, lower alkyl,
lower alkoxy, said phenyl, lower alkyl, and lower alkoxy being
optionally substituted; or a pharmaceutically acceptable salt
thereof or a prodrug thereof, and a pharmaceutical carrier.
5. A method of inhibiting osteoarthritis, hypocalcemia,
hypercalcemia, Paget's disease, osteomalacia, osteohalisteresis,
multiple myeloma or other forms of cancer having deleterious
effects on bone tissues in a mammal in need thereof, comprising
providing to said mammal an effective amount of a compound of the
formula: ##STR25## wherein R.sup.1 and R.sup.2, are each,
independently, H, halogen, CN, phenyl, or lower alkyl; R.sup.3,
R.sup.4, R.sup.5 and R.sup.6, are each independently, H, OH,
halogen, CN, phenyl, lower alkyl, or lower alkoxy; R.sup.8 are
each, independently, H, --C(O)R.sup.9, or lower alkyl; and R.sup.9
is lower alkyl; or a pharmaceutically acceptable salt thereof or a
prodrug thereof.
6. A method of inhibiting benign or malignant abnormal tissue
growth in a mammal in need thereof, comprising providing to said
mammal an effective amount of a compound of the formula: ##STR26##
wherein R.sup.1 and R.sup.2, are each, independently, H, halogen,
CN, phenyl, or lower alkyl; R.sup.3, R.sup.4, R.sup.5 and R.sup.6,
are each independently, H, OH, halogen, CN, phenyl, lower alkyl, or
lower alkoxy; R.sup.8 are each, independently, H, --C(O)R.sup.9, or
lower alkyl; and R.sup.9 is lower alkyl; or a pharmaceutically
acceptable salt thereof or a prodrug thereof.
7. The method of claim 6 wherein the abnormal tissue growth is
prostatic hypertrophy, uterine leiomyomas, breast cancer,
endometriosis, endometrial cancer, polycystic ovary syndrome,
endometrial polyps, benign breast disease, adenomyosis, ovarian
cancer, melanoma, prostrate cancer, cancers of the colon, or CNS
cancers.
8. A method of lowering cholesterol, triglycerides, Lp(a), or LDL
levels; or inhibiting hypercholesteremia; hyperlipidemia;
cardiovascular disease; atherosclerosis; peripheral vascular
disease; restenosis, or vasospasm; or inhibiting vascular wall
damage from cellular events leading toward immune mediated vascular
damage in a mammal in need thereof, comprising providing to said
mammal an effective amount of a compound of the formula: ##STR27##
wherein R.sup.1 and R.sup.2, are each, independently, H, halogen,
CN, phenyl, or lower alkyl; R.sup.3, R.sup.4, R.sup.5 and R.sup.6,
are each independently, H, OH, halogen, CN, phenyl, lower alkyl, or
lower alkoxy; R.sup.8 are each, independently, H, --C(O)R.sup.9, or
lower alkyl; and R.sup.9 is lower alkyl; or a pharmaceutically
acceptable salt thereof or a prodrug thereof.
9. A method of inhibiting free radical induced disease states in a
mammal in need thereof, comprising providing to said mammal an
effective amount of a compound of the formula: ##STR28## wherein
R.sup.1 and R.sup.2, are each, independently, H, halogen, CN,
phenyl, or lower alkyl; R.sup.3, R.sup.4, R.sup.5 and R.sup.6, are
each independently, H, OH, halogen, CN, phenyl, lower alkyl, or
lower alkoxy; R.sup.8 are each, independently, H, --C(O)R.sup.9, or
lower alkyl; and R.sup.9 is lower alkyl; or a pharmaceutically
acceptable salt thereof or a prodrug thereof.
10. A method of providing cognition enhancement or neuroprotection;
or treating or inhibiting senile dementias, Alzheimer's disease,
cognitive decline, or neurodegenerative disorders in a mammal in
need thereof, comprising providing to said mammal an effective
amount of a compound of the formula: ##STR29## wherein R.sup.1 and
R.sup.2, are each, independently, H, halogen, CN, phenyl, or lower
alkyl; R.sup.3, R.sup.4, R.sup.5 and R.sup.6, are each
independently, H, OH, halogen, CN, phenyl, lower alkyl, or lower
alkoxy; R.sup.8 are each, independently, H, --C(O)R.sup.9, or lower
alkyl; and R.sup.9 is lower alkyl; or a pharmaceutically acceptable
salt thereof or a prodrug thereof.
11. A method of inhibiting inflammatory bowel disease, ulcerative
proctitis, Crohn's disease, colitis, hot flashes, vaginal or vulvar
atrophy, atrophic vaginitis, vaginal dryness, pruritus,
dyspareunia, dysuria, frequent urination, urinary incontinence,
urinary tract infections, vasomotor symptoms; male pattern
baldness; skin atrophy; acne; type II diabetes; dysfunctional
uterine bleeding; or infertility in a mammal in need thereof,
comprising providing to said mammal an effective amount of a
compound of the formula: ##STR30## wherein R.sup.1 and R.sup.2, are
each, independently, H, halogen, CN, phenyl, or lower alkyl;
R.sup.3, R.sup.4, R.sup.5 and R.sup.6, are each independently, H,
OH, halogen, CN, phenyl, lower alkyl, or lower alkoxy; R.sup.8 are
each, independently, H, --C(O)R.sup.9, or lower alkyl; and R.sup.9
is lower alkyl; or a pharmaceutically acceptable salt thereof or a
prodrug thereof.
12. A method of inhibiting leukemia, endometrial ablations, chronic
renal or hepatic disease or coagulation diseases or disorders in a
mammal in need thereof, comprising providing to said mammal an
effective amount of a compound of the formula: ##STR31## wherein
R.sup.1 and R.sup.2, are each, independently, H, halogen, CN,
phenyl, or lower alkyl; R.sup.3, R.sup.4, R.sup.5 and R.sup.6, are
each independently, H, OH, halogen, CN, phenyl, lower alkyl, or
lower alkoxy; R.sup.8 are each, independently, H, --C(O)R.sup.9, or
lower alkyl; and R.sup.9 is lower alkyl; or a pharmaceutically
acceptable salt thereof or a prodrug thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S.
application Ser. No. 10/835,228 filed Apr. 29, 2004. The
application also claims benefit to U.S. Provisional Application
Ser. No. 60/467,394, filed May 2, 2003. Both applications are
incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
[0002] This invention relates to novel
4'-hydroxy-biphenyl-carbaldehyde oxime derivatives, their uses as
estrogenic agents, and methods of their preparation.
BACKGROUND OF THE INVENTION
[0003] The pleiotropic effects of estrogens in mammalian tissues
have been well documented, and it is now appreciated that estrogens
affect many organ systems [Mendelsohn and Karas, New England
Journal of Medicine 340: 1801-1811 (1999), Epperson, et al.,
Psychosomatic Medicine 61: 676-697 (1999), Crandall, Journal of
Womens Health & Gender Based Medicine 8: 1155-1166 (1999), Monk
and Brodaty, Dementia & Geriatric Cognitive Disorders 11: 1-10
(2000), Hum and Macrae, Journal of Cerebral Blood Flow &
Metabolism 20: 631-652 (2000), Calvin, Maturitas 34: 195-210
(2000), Finking, et al., Zeitschrift fur Kardiologie 89: 442-453
(2000), Brincat, Maturitas 35: 107-117 (2000), Al-Azzawi,
Postgraduate Medical Journal 77: 292-304 (2001)]. Estrogens can
exert effects on tissues in several ways. Probably, the most well
characterized mechanism of action is their interaction with
estrogen receptors leading to alterations in gene transcription.
Estrogen receptors are ligand-activated transcription factors and
belong to the nuclear hormone receptor superfamily. Other members
of this family include the progesterone, androgen, glucocorticoid
and mineralocorticoid receptors. Upon binding ligand, these
receptors dimerize and can activate gene transcription either by
directly binding to specific sequences on DNA (known as response
elements) or by interacting with other transcription factors (such
as AP1), which in turn bind directly to specific DNA sequences
[Moggs and Orphanides, EMBO Reports 2: 775-781 (2001), Hall, et
al., Journal of Biological Chemistry 276: 36869-36872 (2001),
McDonnell, Principles Of Molecular Regulation. p351-361 (2000)]. A
class of "coregulatory" proteins can also interact with the
ligand-bound receptor and further modulate its transcriptional
activity [McKenna, et al., Endocrine Reviews 20: 321-344 (1999)].
It has also been shown that estrogen receptors can suppress
NF.kappa.B-mediated transcription in both a ligand-dependent and
independent manner [Quaedackers, et al., Endocrinology 142:
1156-1166 (2001), Bhat, et al., Journal of Steroid Biochemistry
& Molecular Biology 67: 233-240 (1998), Pelzer, et al.,
Biochemical & Biophysical Research Communications 286: 1153-7
(2001)].
[0004] Estrogen receptors can also be activated by phosphorylation.
This phosphorylation is mediated by growth factors such as EGF and
causes changes in gene transcription in the absence of ligand
[Moggs and Orphanides, EMBO Reports 2: 775-781 (2001), Hall, et
al., Journal of Biological Chemistry 276: 36869-36872 (2001)].
[0005] A less well-characterized means by which estrogens can
affect cells is through a so-called membrane receptor. The
existence of such a receptor is controversial, but it has been well
documented that estrogens can elicit very rapid non-genomic
responses from cells. The molecular entity responsible for
transducing these effects has not been definitively isolated, but
there is evidence to suggest it is at least related to the nuclear
forms of the estrogen receptors [Levin, Journal of Applied
Physiology 91: 1860-1867 (2001), Levin, Trends in Endocrinology
& Metabolism 10: 374-377 (1999)].
[0006] Two estrogen receptors have been discovered to date. The
first estrogen receptor was cloned about 15 years ago and is now
referred to as ER.alpha. [Green, et al., Nature 320: 134-9 (1986)].
The second was found comparatively recently and is called ER.beta.
[Kuiper, et al., Proceedings of the National Academy of Sciences of
the United States of America 93: 5925-5930 (1996)]. Early work on
ER.beta. focused on defining its affinity for a variety of ligands
and, indeed, some differences with ER.alpha. were seen. The tissue
distribution of ER.beta. has been well mapped in the rodent and it
is not coincident with ER.alpha.. Tissues such as the mouse and rat
uterus express predominantly ER.alpha., whereas the mouse and rat
lung express predominantly ER.beta.[Couse, et al., Endocrinology
138: 4613-4621 (1997), Kuiper, et al., Endocrinology 138: 863-870
(1997)]. Even within the same organ, the distribution of ER.alpha.
and ER.alpha. can be compartmentalized. For example, in the mouse
ovary, ER.alpha. is highly expressed in the granulosa cells and
ER.alpha. is restricted to the thecal and stromal cells [Sar and
Welsch, Endocrinology 140: 963-971 (1999), Fitzpatrick, et al.,
Endocrinology 140: 2581-2591 (1999)]. However, there are examples
where the receptors are coexpressed and there is evidence from in
vitro studies that ER.alpha. and ER.alpha. can form heterodimers
[Cowley, et al., Journal of Biological Chemistry 272: 19858-19862
(1997)].
[0007] The most potent endogenous estrogen is 17.beta.-estradiol. A
large number of compounds have been described that either mimic or
block the activity of 17.beta.-estradiol. Compounds having roughly
the same biological effects as 17.beta.-estradiol are referred to
as "estrogen receptor agonists". Those which block the effects of
17.beta.-estradiol, when given in combination with it, are called
"estrogen receptor antagonists". In reality, there is a continuum
between estrogen receptor agonist and estrogen receptor antagonist
activity and some compounds behave as estrogen receptor agonists in
some tissues but estrogen receptor antagonists in others. These
compounds with mixed activity are called selective estrogen
receptor modulators (SERMS) and are therapeutically useful agents
(e.g. EVISTA) [McDonnell, Journal of the Society for Gynecologic
Investigation 7: S10-S15 (2000), Goldstein, et al., Human
Reproduction Update 6: 212-224 (2000)]. The precise reason why the
same compound can have cell-specific effects has not been
elucidated, but the differences in receptor conformation and/or in
the milieu of coregulatory proteins have been suggested.
[0008] It has been known for some time that estrogen receptors
adopt different conformations when binding ligands. However, the
consequence and subtlety of these changes only recently has been
revealed. The three dimensional structures of ER.alpha. and
ER.beta. have been solved by co-crystallization with various
ligands and clearly show the repositioning of helix 12 in the
presence of an estrogen receptor antagonist, which sterically
hinders the protein sequences required for receptor-coregulatory
protein interaction [Pike, et al., Embo 18: 4608-4618 (1999),
Shiau, et al., Cell 95: 927-937 (1998)]. In addition, the technique
of phage display has been used to identify peptides that interact
with estrogen receptors in the presence of different ligands
[Paige, et al., Proceedings of the National Academy of Sciences of
the United States of America 96: 3999-4004 (1999)]. For example, a
peptide was identified that distinguished between ER.alpha. bound
to the full estrogen receptor agonists 17.beta.-estradiol and
diethylstilbesterol. A different peptide was shown to distinguish
between clomiphene bound to ER.alpha. and ER.beta.. These data
indicate that each ligand potentially places the receptor in a
unique and unpredictable conformation that is likely to have
distinct biological activities.
[0009] As mentioned above, estrogens affect a panoply of biological
processes. In addition, where gender differences have been
described (e.g. disease frequencies, responses to challenge, etc),
it is possible that the explanation involves the difference in
estrogen levels between males and females.
SUMMARY OF THE INVENTION
[0010] The present invention relates to a compound of the formula:
##STR2## where
[0011] R.sup.1 and R.sup.2, are each, independently, H, halogen,
CN, phenyl, or lower alkyl;
[0012] R.sup.3, R.sup.4, R.sup.5 and R.sup.6, are each
independently, H, OH, halogen, CN, phenyl, lower alkyl, or lower
alkoxy;
[0013] R.sup.8 are each, independently, H, --C(O)R.sup.9, or lower
alkyl; and
[0014] R.sup.9 is lower alkyl;
[0015] or a pharmaceutically acceptable salt thereof or a prodrug
thereof. In one preferred embodiment, R.sup.8 is H.
[0016] In another aspect, the invention relates to a compound of
the formula: ##STR3##
[0017] In yet another aspect, the invention is directed to a
compound of the formula: ##STR4##
[0018] In a further aspect, the invention is drawn to a compound of
the formula ##STR5## where R.sup.1 is OH or lower alkoxy; and
R.sup.5, R.sup.6, and R.sup.7 are each, independently, H, OH,
halogen, CN, phenyl, lower alkyl, lower alkoxy, said phenyl, lower
alkyl, and lower alkoxy being optionally substituted; or a
pharmaceutically acceptable salt thereof or a prodrug thereof.
[0019] In another aspect, the invention is drawn to a
pharmaceutical composition that comprises one or more of compound
of the invention and a pharmaceutically acceptable carrier.
[0020] In yet other aspects, the invention is directed to use of
the compounds of the invention in the treatment or prevention of
diseases such as inflammatory bowel diseases.
DETAILED DESCRIPTION OF THE INVENTION
[0021] This invention provides novel
4'-hydroxy-biphenyl-carbaldehyde oxime derivatives. These
compounds, which preferably act as estrogenic agents, are useful
for the treatment and prevention of diseases such as inflammatory
bowel diseases (including Crohn's disease and colitis). In one
aspect, the invention is directed to compounds of the formula:
##STR6## where
[0022] R.sup.1 and R.sup.2, are each, independently, H, halogen,
CN, phenyl, or lower alkyl;
[0023] R.sup.3, R.sup.4, R.sup.5 and R.sup.6, are each
independently, H, OH, halogen, CN, phenyl, lower alkyl, or lower
alkoxy;
[0024] R.sup.8 are each, independently, H, --C(O)R.sup.9, or lower
alkyl; and
[0025] R.sup.9 is lower alkyl;
[0026] or a pharmaceutically acceptable salt thereof or a prodrug
thereof. In one preferred embodiment, R.sup.8 is H.
[0027] Compounds according to the invention can be: ##STR7##
[0028] In certain aspects, R.sup.3, R.sup.5, and R.sup.6 are each
independently H, Cl, F, methyl, or methoxy and R.sup.2 is H, Cl, F,
or methyl. In another aspect, the invention is directed to
compounds of the formula: ##STR8##
[0029] In certain aspects, R.sup.3, R.sup.5, and R.sup.6 are each
independently H, Cl, F, methyl, or methoxy and R.sup.2 is H, Cl, F,
or methyl.
[0030] In yet another aspect, the invention is drawn to compounds
of the formula ##STR9## where R.sup.1 is OH or lower alkoxy; and
R.sup.5, R.sup.6, and R.sup.7 are each, independently, H, OH,
halogen, CN, phenyl, lower alkyl, lower alkoxy; or a
pharmaceutically acceptable salt thereof or a prodrug thereof. In
certain embodiments, R.sup.5, R.sup.6, and R.sup.7 are each,
independently, H, Me, Cl, F, or methoxy.
[0031] Pharmaceutically acceptable salts can be formed from organic
and inorganic acids, for example, acetic, propionic, lactic,
citric, tartaric, succinic, fumaric, maleic, malonic, mandelic,
malic, phthalic, hydrochloric, hydrobromic, phosphoric, nitric,
sulfuric, methanesulfonic, napthalenesulfonic, benzenesulfonic,
toluenesulfonic, camphorsulfonic, and similarly known acceptable
aids when a compound of this invention contains a basic moiety.
Salts may also be formed from organic and inorganic bases, such as
alkali metal salts (for example, sodium, lithium, or potassium)
alkaline earth metal salts, ammonium salts, alkylammonium salts
containing 1-6 carbon atoms or dialkylammonium salts containing 1-6
carbon atoms in each alkyl group, and trialkylammonium salts
containing 1-6 carbon atoms in each alkyl group, when a compound of
this invention contains an acidic moiety.
[0032] The term "alkyl", as used herein, whether used alone or as
part of another group, refers to a substituted or unsubstituted
aliphatic hydrocarbon chain and includes, but is not limited to,
straight and branched chains containing from 1 to 12 carbon atoms,
preferably 1 to 6 carbon atoms, unless explicitly specified
otherwise. For example, methyl, ethyl, propyl, isopropyl, butyl,
i-butyl and t-butyl are encompassed by the term "alkyl."
Specifically included within the definition of "alkyl" are those
aliphatic hydrocarbon chains that are optionally substituted.
[0033] The carbon number as used in the definitions herein refers
to carbon backbone and carbon branching, but does not include
carbon atoms of the substituents, such as alkoxy substitutions and
the like.
[0034] The term "alkenyl", as used herein, whether used alone or as
part of another group, refers to a substituted or unsubstituted
aliphatic hydrocarbon chain and includes, but is not limited to,
straight and branched chains having 2 to 8 carbon atoms and
containing at least one double bond. Preferably, the alkenyl moiety
has 1 or 2 double bonds. Such alkenyl moieties may exist in the E
or Z conformations and the compounds of this invention include both
conformations. Specifically included within the definition of
"alkenyl" are those aliphatic hydrocarbon chains that are
optionally substituted. Heteroatoms, such as O, S or N--R.sub.1,
attached to an alkenyl should not be attached to a carbon atom that
is bonded to a double bond.
[0035] The term "phenyl", as used herein, whether used alone or as
part of another group, refers to a substituted or unsubstituted
phenyl group.
[0036] An optionally substituted alkyl, alkenyl, and phenyl may be
substituted with one or more substituents. Suitable optionally
substituents may be selected independently from nitro, cyano,
--N(R.sub.11)(R.sub.12), halo, hydroxy, carboxy, alkyl, alkenyl,
alkynyl, cycloalkyl, aryl, heteroaryl, alkoxy, aryloxy,
heteroaryloxy, alkylalkoxy, alkoxycarbonyl, alkoxyalkoxy,
perfluoroalkyl, perfluoroalkoxy, arylalkyl, alkylaryl,
hydroxyalkyl, alkoxyalkyl, alkylthio,
--S(O).sub.2--N(R.sub.11)(R.sub.12),
--C(.dbd.O)--N(R.sub.11)(R.sub.12), (R.sub.11)(R.sub.12)N-alkyl,
(R.sub.11)(R.sub.12)N-alkoxyalkyl,
(R.sub.11)(R.sub.12)N-alkylaryloxyalkyl, --S(O).sub.s-aryl (where
s=0-2) or --S(O).sub.s-heteroaryl (where s=0-2). In certain
embodiments of the invention, preferred substitutents for alkyl,
alkenyl, alkynyl and cycloalkyl include nitro, cyano,
--N(R.sub.11)(R.sub.12), halo, hydroxyl, aryl, heteroaryl, alkoxy,
alkoxyalkyl, and alkoxycarbonyl. In certain embodiments of the
invention, preferred substituents for aryl and heteroaryl include
--N(R.sub.11)(R.sub.12), alkyl, halo, perfluoroalkyl,
perfluoroalkoxy, arylalkyl and alkylaryl.
[0037] When alkyl or alkenyl moieties are substituted, for example,
they may typically be mono-, di-, tri- or persubstituted. Examples
for a halogen substituent include 1-bromo vinyl, 1-fluoro vinyl,
1,2-difluoro vinyl, 2,2-difluorovinyl, 1,2,2-trifluorovinyl,
1,2-dibromo ethane, 1,2 difluoro ethane, 1-fluoro-2-bromo ethane,
CF.sub.2CF.sub.3, CF.sub.2CF.sub.2CF.sub.3, and the like.
[0038] The term halogen includes bromine, chlorine, fluorine, and
iodine.
[0039] The term "lower alkyl" refers to an alkyl group having 1 to
6 carbon atoms, in some embodiments 1 to 3 carbon atoms are
preferred.
[0040] The term "lower alkoxy," as used herein, refers to the group
R--O--where R is an alkyl group of 1 to 6 carbon atoms, in some
embodiments 1 to 3 carbon atoms are preferred.
[0041] As used in accordance with this invention, the term
"providing," with respect to providing a compound or substance
covered by this invention, means either directly administering such
a compound or substance, or administering a prodrug, derivative, or
analog which will form the equivalent amount of the compound or
substance within the body.
[0042] The compounds of this invention can be used as estrogen
receptor modulators useful in the treatment or inhibition of
conditions, disorders, or disease states that are at least
partially mediated by an estrogen deficiency or excess, or which
may be treated or inhibited through the use of an estrogenic agent.
The compounds of this invention are particularly useful in treating
a peri-menopausal, menopausal, or postmenopausal patient in which
the levels of endogenous estrogens produced are greatly diminished.
Menopause is generally defined as the last natural menstrual period
and is characterized by the cessation of ovarian function, leading
to the substantial diminution of circulating estrogen in the
bloodstream. As used herein, menopause also includes conditions of
decreased estrogen production that may be surgically, chemically,
or be caused by a disease state which leads to premature diminution
or cessation of ovarian function.
[0043] Accordingly, the compounds of this invention are useful in
treating or inhibiting osteoporosis and in the inhibition of bone
demineralization, which may result from an imbalance in a
individual's formation of new bone tissues and the resorption of
older tissues, leading to a net loss of bone. Such bone depletion
results in a range of individuals, particularly in post-menopausal
women, women who have undergone bilateral oophorectomy, those
receiving or who have received extended corticosteroid therapies,
those experiencing gonadal dysgenesis, and those suffering from
Cushing's syndrome. Special needs for bone, including teeth and
oral bone, replacement can also be addressed using these compounds
in individuals with bone fractures, defective bone structures, and
those receiving bone-related surgeries and/or the implantation of
prosthesis. In addition to those problems described above, these
compounds can be used in treatment or inhibition for
osteoarthritis, hypocalcemia, hypercalcemia, Paget's disease,
osteomalacia, osteohalisteresis, multiple myeloma and other forms
of cancer having deleterious effects on bone tissues.
[0044] The compounds of this invention are also useful in treating
or inhibiting benign or malignant abnormal tissue growth, including
prostatic hypertrophy, uterine leiomyomas, breast cancer,
endometriosis, endometrial cancer, polycystic ovary syndrome,
endometrial polyps, benign breast disease, adenomyosis, ovarian
cancer, melanoma, prostrate cancer, cancers of the colon, CNS
cancers, such as glioma or astioblastomia.
[0045] The compounds of this invention are cardioprotective and
they are useful in lowering cholesterol, triglycerides, Lp(a), and
LDL levels; inhibiting or treating hypercholesteremia;
hyperlipidemia; cardiovascular disease; atherosclerosis; peripheral
vascular disease; restenosis, and vasospasm; and inhibiting
vascular wall damage from cellular events leading toward immune
mediated vascular damage. These cardiovascular protective
properties are of great importance when treating postmenopausal
patients with estrogens to inhibit osteoporosis and in the male
when estrogen therapy is indicated.
[0046] The compounds of this invention are also antioxidants, and
are therefore useful in treating or inhibiting free radical induced
disease states. Specific situations in which antioxidant therapy is
indicated to be warranted are with cancers, central nervous system
disorders, Alzheimer's disease, bone disease, aging, inflammatory
disorders, peripheral vascular disease, rheumatoid arthritis,
autoimmune diseases, respiratory distress, emphysema, prevention of
reperfusion injury, viral hepatitis, chronic active hepatitis,
tuberculosis, psoriasis, systemic lupus erythematosus, adult
respiratory distress syndrome, central nervous system trauma and
stroke.
[0047] The compounds of this invention are also useful in providing
cognition enhancement, and in treating or inhibiting senile
dementias, Alzheimer's disease, cognitive decline,
neurodegenerative disorders, providing neuroprotection or cognition
enhancement.
[0048] The compounds of this invention are also useful in treating
or inhibiting inflammatory bowel disease, ulcerative proctitis,
Crohn's disease, and colitis; menopausal related conditions, such
as vasomotor symptoms including hot flushes, vaginal or vulvar
atrophy, atrophic vaginitis, vaginal dryness, pruritus,
dyspareunia, dysuria, frequent urination, urinary incontinence,
urinary tract infections, vasomotor symptoms, including hot
flushes, myalgia, arthralgia, insomnia, irritability, and the like;
male pattern baldness; skin atrophy; acne; type TI diabetes;
dysfunctional uterine bleeding; and infertility.
[0049] The compounds of this invention are useful in disease states
where amenorrhea is advantageous, such as leukemia, endometrial
ablations, chronic renal or hepatic disease or coagulation diseases
or disorders.
[0050] The compounds of this invention can be used as a
contraceptive agent, particularly when combined with a
progestin.
[0051] When administered for the treatment or inhibition of a
particular disease state or disorder, it is understood that the
effective dosage may vary depending upon the particular compound
utilized, the mode of administration, the condition, and severity
thereof, of the condition being treated, as well as the various
physical factors related to the individual being treated. Effective
administration of the compounds of this invention may be given at
an oral dose of from about 0.1 mg/day to about 1,000 mg/day.
Preferably, administration will be from about 10 mg/day to about
600 mg/day, more preferably from about 50 mg/day to about 600
mg/day, in a single dose or in two or more divided doses. The
projected daily dosages are expected to vary with route of
administration.
[0052] Such doses may be administered in any manner useful in
directing the active compounds herein to the recipient's
bloodstream, including orally, via implants, parenterally
(including intravenous, intraperitoneal and subcutaneous
injections), rectally, intranasally, vaginally, and
transdermally.
[0053] Oral formulations containing the active compounds of this
invention may comprise any conventionally used oral forms,
including tablets, capsules, buccal forms, troches, lozenges and
oral liquids, suspensions or solutions. Capsules may contain
mixtures of the active compound(s) with inert fillers and/or
diluents such as the pharmaceutically acceptable starches (e.g.
corn, potato or tapioca starch), sugars, artificial sweetening
agents, powdered celluloses, such as crystalline and
microcrystalline celluloses, flours, gelatins, gums, etc. Useful
tablet formulations may be made by conventional compression, wet
granulation or dry granulation methods and utilize pharmaceutically
acceptable diluents, binding agents, lubricants, disintegrants,
surface modifying agents (including surfactants), suspending or
stabilizing agents, including, but not limited to, magnesium
stearate, stearic acid, talc, sodium lauryl sulfate,
microcrystalline cellulose, carboxymethylcellulose calcium,
polyvinylpyrrolidone, gelatin, alginic acid, acacia gum, xanthan
gum, sodium citrate, complex silicates, calcium carbonate, glycine,
dextrin, sucrose, sorbitol, dicalcium phosphate, calcium sulfate,
lactose, kaolin, mannitol, sodium chloride, talc, dry starches and
powdered sugar. Preferred surface modifying agents include nonionic
and anionic surface modifying agents. Representative examples of
surface modifying agents include, but are not limited to, poloxamer
188, benzalkonium chloride, calcium stearate, cetostearl alcohol,
cetomacrogol emulsifying wax, sorbitan esters, colloidol silicon
dioxide, phosphates, sodium dodecylsulfate, magnesium aluminum
silicate, and triethanolamine. Oral formulations herein may utilize
standard delay or time release formulations to alter the absorption
of the active compound(s). The oral formulation may also consist of
administering the active ingredient in water or a fruit juice,
containing appropriate solubilizers or emulsifiers as needed.
[0054] In some cases it may be desirable to administer the
compounds directly to the airways in the form of an aerosol.
[0055] The compounds of this invention may also be administered
parenterally or intraperitoneally. Solutions or suspensions of
these active compounds as a free base or pharmacologically
acceptable salt can be prepared in water suitably mixed with a
surfactant such as hydroxy-propylcellulose. Dispersions can also be
prepared in glycerol, liquid polyethylene glycols and mixtures
thereof in oils. Under ordinary conditions of storage and use,
these preparation contain a preservative to prevent the growth of
microorganisms.
[0056] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions and sterile powders for
the extemporaneous preparation of sterile injectable solutions or
dispersions. In all cases, the form must be sterile and must be
fluid to the extent that easy syringability exists. It must be
stable under the conditions of manufacture and storage and must be
preserved against the contaminating action of microorganisms such
as bacteria and fungi. The carrier can be a solvent or dispersion
medium containing, for example, water, ethanol, polyol (e.g.,
glycerol, propylene glycol and liquid polyethylene glycol),
suitable mixtures thereof, and vegetable oils.
[0057] For the purposes of this disclosure, transdermal
administrations are understood to include all administrations
across the surface of the body and the inner linings of bodily
passages including epithelial and mucosal tissues. Such
administrations may be carried out using the present compounds, or
pharmaceutically acceptable salts thereof, in lotions, creams,
foams, patches, suspensions, solutions, and suppositories (rectal
and vaginal).
[0058] Transdermal administration may be accomplished through the
use of a transdermal patch containing the active compound and a
carrier that is inert to the active compound, is non toxic to the
skin, and allows delivery of the agent for systemic absorption into
the blood stream via the skin. The carrier may take any number of
forms such as creams and ointments, pastes, gels, and occlusive
devices. The creams and ointments may be viscous liquid or
semisolid emulsions of either the oil-in-water or water-in-oil
type. Pastes comprised of absorptive powders dispersed in petroleum
or hydrophilic petroleum containing the active ingredient may also
be suitable. A variety of occlusive devices may be used to release
the active ingredient into the blood stream such as a
semi-permeable membrane covering a reservoir containing the active
ingredient with or without a carrier, or a matrix containing the
active ingredient. Other occlusive devices are known in the
literature.
[0059] Suppository formulations may be made from traditional
materials, including cocoa butter, with or without the addition of
waxes to alter the suppository's melting point, and glycerin. Water
soluble suppository bases, such as polyethylene glycols of various
molecular weights, may also be used.
[0060] The reagents used in the preparation of the compounds of
this invention can be either commercially obtained or can be
prepared by standard procedures described in the literature.
[0061] The preparation of several representative examples of this
invention are described in the following Schemes 1-11. ##STR10##
##STR11## ##STR12## ##STR13## ##STR14## ##STR15## ##STR16##
##STR17## ##STR18## ##STR19## ##STR20## ##STR21##
EXAMPLE 1
4-tert-Butyl-dimethylsilyoxyphenylboronic acid
[0062] Method A. To a solution of
(4-bromo-phenoxy)-tert-butyl-dimethylsilane (20 mL, 23.48 g, 0.082
moles) in tetrahydrofuran (200 mL) at -78.degree. C. was slowly
added n-butyl lithium (39.2 mL of 2.5 M solution in hexane, 0.098
moles) under N.sub.2 with stirring over a few minutes. The solution
was stirred for 1 hour and triisopropyl borate (66.2 mL, 54.0 g,
0.29 moles) was added by syringe at -78.degree. C. The solution was
stirred for 1 hr at -78.degree. C. and then allowed to warm to room
temperature overnight. The reaction was cooled to 0.degree. C. and
water (20 mL) and 2 N HCl (20 mL) were added into the reaction
mixture. Then the whole mixture was stirred with of 2 N HCl (360
mL) for 10 minutes. The mixture was extracted with ethyl acetate
(3.times.250 mL). The combined organic layers were concentrated to
a volume of about 50 mL. Crystallization was induced with cold
hexane, and the solid product was collected by filtration and dried
under vacuum to yield 14.5 g (70%) of the title compound as a white
solid: .sup.1H NMR (DMSO-d.sub.6): .delta. 0.19 (6H, s), 0.94 (9H,
s), 6.80 (2H, d, J=8.4 Hz), 7.69 (2H, d, J=8.36 Hz), 7.87 (2H,
s).
EXAMPLE 2
4'-Hydroxy-3-methyl[1,1'-biphenyl]-4-carbonitrile
[0063] Method B. A mixture of 4-bromo-2-methylbenzonitrile (1.8 g,
9.18 mmol), 4-tert-butyl-dimethylsilyoxyphenylboronic acid (3.0 g,
11.9 mmol), sodium carbonate (13.8 mL of 2 M aqueous solution, 27.5
mmol), tetrakis(triphenylphosphine)palladium (0.53 g, 0.46 mmol),
and ethylene glycol dimethyl ether (70 mL) were heated to reflux
overnight. The mixture was cooled to room temperature and poured
into water, then extracted with ethyl acetate (3.times.), washed
with brine, dried over sodium sulfate, filtered, and the solvent
evaporated. Purification by silica chromatography (15%-25% ethyl
acetate-hexane) to yield 1.81 g (94%) of the title compound as a
yellowish solid: mp 177-178.degree. C.; .sup.1H NMR (DMSO-d.sub.6):
.delta. 2.52 (3H, s), 6.88 (2H, d, J=8.50 Hz), 7.60 (3H, d, J=8.49
Hz), 7.71 (1H, s), 7.77 (2H, d, J=8.12 Hz), 9.79 (1H, s); IR 2220
cm.sup.-1; MS (ESI) m/z 208 (M-H).sup.-.
[0064] Anal. for C.sub.14H.sub.11NO:
[0065] Calc'd: C, 80.36; H, 5.30; N, 6.69.
[0066] Found: C, 79.91; H, 5.27; N, 6.57.
EXAMPLE 3
4'-Hydroxy-3-methyl[1,1'-biphenyl]-4-carbaldehyde
[0067] A solution of
4'-hydroxy-3-methyl[1,1'-biphenyl]-4-carbonitrile (500 mg, 2.39
mmol) in dry toluene was cooled to -78.degree. C. and
diisobutylaluminum hydride (4.0 mL of 1.5 M solution in toluene,
5.98 mmol) was added all at once. The reaction mixture was allowed
to warm to room temperature over a period of 3.5 h. Methanol (1.2
mL) was added followed by water (1.2 mL) at 0.degree. C. and the
mixture was stirred at room temperature for 20 min. 1 N HCl
solution was added with stirring until pH <7. The mixture was
extracted with ethyl acetate (3.times.), washed with brine, dried
over sodium sulfate, filtered, and the solvent evaporated.
Purification by silica chromatography (15%-25% ethyl
acetate-hexane) to yield 459 mg (91%) of the title compound as a
white solid: mp 161-162.degree. C.; .sup.1H NMR (DMSO-d.sub.6):
.delta. 2.67 (3H, s), 6.88 (2H, d, J=8.41 Hz), 7.59-7.65 (4H, m),
7.85 (1H, d, J=8.03 Hz), 9.78 (1H, s), 10.22 (1H, s); IR 1680
cm.sup.-1; MS (ESI) m/z 211 (M-H).sup.-.
[0068] Anal. for C.sub.14H.sub.12O.sub.2:
[0069] Calc'd: C, 79.23; H, 5.70.
[0070] Found: C, 78.79; H, 5.90.
EXAMPLE 4
4'-{[tert-Butyl(dimethyl)silyl]oxy}-1,1'-biphenyl-4-carbaldehyde
[0071] The title compound was prepared by reacting
4-bromobenzaldehyde (730 mg, 3.97 mmol) with
4-tert-butyl-dimethylsilyoxyphenylboronic acid (1.3 g, 5.16 mmol)
according to Method B to yield 600 mg (48%) of white crystal:
.sup.1H NMR (DMSO-d.sub.6): .delta. 0.24 (6H, s), 1.01 (9H, s),
6.94 (2H, d, J=8.54 Hz), 7.53 (2H, d, J=8.56 Hz), 7.72 (2H, d,
J=8.19 Hz), 7.93 (2H, d, 8.20 Hz), 10.04 (1H, s).
EXAMPLE 5
4'-Hydroxy[1,1'-biphenyl]-4-carbaldehyde
[0072] A mixture of
4'-{[tert-butyl(dimethyl)silyl]oxy}-1,1'-biphenyl-4-carbaldehyde
(320 mg, 1.03 mmol), anhydrous KF (120 mg, 2.06 mmol) and 48%
aqueous HBr (35 ul, 0.31 mmol) in 6 mL dry DMF was stirred at room
temperature under N.sub.2 for 1 h. TLC indicated starting material
present and therefore more 48% aqueous HBr (35 uL, 0.3 mmol) was
added into the reaction and the mixture was continued to stir for
1.5 h. The mixture was then poured, with cooling, into 1 N aqueous
HCl (30 mL). The aqueous mixture was extracted with EtOAc
(3.times.). The combined extracts were washed with saturated NaCl
solution, and dried over Na.sub.2SO.sub.4. The solvent was
evaporated under reduced pressure and the product (100%) which was
used directly in the next step. .sup.1H NMR (DMSO-d.sub.6): .delta.
6.89 (2H, d, J=8.43 Hz), 7.63 (2H, d, J=8.46 Hz), 7.83 (2H, d,
J=8.16 Hz), 7.94 (2H, d, J=8.02 Hz), 9.79 (1H, s), 10.01 (1H,
s).
EXAMPLE 6
Trifluoro-methanesulfonic acid 3-chloro-4-formyl-phenyl ester
[0073] Method D. To a solution of 2-chloro-4-hydroxy benzaldehyde
(1.31 g, 8.4 mmol) and pyridine (1.1 mL, 13.4 mmol) in 80 mL of
dichloromethane at 0.degree. C. was added trifluoromethanesulfonic
anhydride (1.84 mL, 3.08 g, 10.9 mmol). The solution was allowed to
slowly warm to room temperature and stirred for 2.5 h. The solution
was cooled to 0.degree. C. and stirred with ice water to decompose
any excess anhydride. The mixture was made slightly basic by the
addition of saturated sodium bicarbonate solution. The resulting
layers were separated and the aqueous layer was extracted with
dichloromethane (3.times.100 mL). The combined organic layers were
washed with brine, dried over sodium sulfate, filtered, and the
solvent removed under vacuum to afford a orange oil which was
purified by silica chromatography (5% ethyl acetate-hexanes) to
yield 1.83 g (76%) of the title compound as a clear, colorless oil:
.sup.1H NMR (DMSO-d.sub.6) .delta. 7.73 (1H, dd, J=2.35 Hz, J=8.64
Hz), 8.04-8.07 (2H, m), 10.30 (1H, s).
EXAMPLE 7
Trifluoro-methanesulfonic acid 2-fluoro-4-formyl-phenyl ester
[0074] The title compound was prepared by reacting
3-fluoro-4-hydroxybenzaldehyde (1.2 g, 8.56 mmol) with
trifluoromethanesulfonic anhydride (1.87 mL, 3.14 g, 11.1 mmol)
according to Method D to yield a yellow oil which was used directly
in the next step without purification: .sup.1H NMR (CDCl.sub.3):
.delta. 7.53-7.58 (1H, m), 7.77-7.85 (2H, m), 10.01 (1H, d, J=1.45
Hz).
EXAMPLE 8
4'-{[Tert-butyl(dimethyl)silyl]oxy}-3-chloro-1,1'-biphenyl-4-carbaldehyde
[0075] The title compound was prepared by reacting
trifluoro-methanesulfonic acid 3-chloro-4-formyl-phenyl ester (1.78
g, 6.18 mmol) with 4-tert-butyl-dimethylsilyoxyphenylboronic acid
(2.03 g, 8.03 mmol) according to Method B to yield 0.92 g (43%) of
white solid: mp 38-39.degree. C.; .sup.1H NMR (DMDO-d.sub.6):
.delta. 0.23 (6H, s), 0.97 (9H, s), 6.98 (2H, d, J=8.79 Hz), 7.74
(2H, d, J=8.79 Hz), 7.81 (1H, d, J=7.81 Hz), 7.89 (1H, d, J=1.95
Hz), 7.91 (1H, d, J=7.81 Hz), 10.34 (1H, s); MS (ESI) m/z 231/233
(M-H).sup.- (deprotected product ion).
[0076] Anal. for C.sub.19H.sub.23ClO.sub.2Si:
[0077] Calc'd: C, 65.78; H, 6.68.
[0078] Found: C, 65.59; H, 6.60.
EXAMPLES 9 AND 10
[0079] Trifluoro-methanesulfonic acid 2-fluoro-4-formyl-phenyl
ester (2.1 g, 7.72 mmol) were reacted with
4-tert-butyl-dimethylsilyoxyphenylboronic acid (2.14 g, 8.49 mmol)
according to Method B to produce the following two compounds:
4'-{[tert-butyl(dimethyl)silyl]oxy}-2-fluoro-1,1'-biphenyl-4-carbaldehyde
[0080] 1.62 g (63%) of waxy yellowish solid: .sup.1H NMR
(Acetone-d.sub.6): .delta. 0.28 (6H, s), 1.02 (9H, s), 7.02-7.06
(2H, m), 7.57-7.60 (2H, m), 7.71-7.78 (2H, m), 7.85 (1H, dd, J=7.91
Hz, J=1.51 Hz), 10.07 (1H, d, J=1.73 Hz); .sup.13C NMR
(Acetone-d.sub.6): .delta. -4.29, 18.80, 26.00, 116.82 (d, J=23.90
Hz), 121.15, 126.69 (d, J=3.25 Hz), 128.42 (d, 1.21 Hz), 131.30 (d,
J=3.48 Hz), 132.20 (d, J=3.44 Hz), 135.27 (d, J=13.61), 138.09 (d,
J=6.64 Hz), 157.23, 160.68 (d, J=248.56 Hz), 191.51; IR 1692
cm.sup.-1; MS (ESI) m/z 331 (M+H).sup.+, 372 (M+H+ ACN).sup.+.
[0081] Anal. for C.sub.19H.sub.23FO.sub.2Si:
[0082] Calc'd: C, 69.06; H, 7.02.
[0083] Found: C, 69.71; H, 7.34.
2-Fluoro-4'-hydroxy-1,1'-biphenyl-4-carbaldehyde
[0084] 0.32 g (19%) of yellowish solid: mp 149-150.degree. C.;
.sup.1H NMR (Acetone-d.sub.6): .delta. 7.08-7.13 (2H, m), 7.62-7.67
(2H, m), 7.80-7.91 (2H, m), 7.94 (1H, dd, J=7.92 Hz, J=1.58 Hz),
8.93 (1H, s), 10.16 (1H, d, J=1.73 Hz); IR 1669 cm.sup.-1; MS (ESI)
m/z 215 (M-H).sup.-1
[0085] Anal. for C.sub.13H.sub.9FO.sub.2:
[0086] Calc'd: C, 72.22; H, 4.20.
[0087] Found: C, 71.38; H, 4.12.
EXAMPLE 11
4'-{[tert-Butyl(dimethyl)silyl]oxy}-3-chloro[1,1'-biphenyl]-4-carbaldehyde
oxime
[0088] The title compound was prepared by reacting
4'-{[tert-butyl(dimethyl)silyl]oxy}-3-chloro-1,1'-biphenyl-4-carbaldehyde
(405 mg, 1.17 mmol) with hydroxylamine hydrochloride (154 mg, 2.22
mmol) according to Method C to yield a yellowish oil which was used
in the next step without purification: MS (ESI) m/z 362/364
(M+H).sup.+.
EXAMPLE 12
4'-{[Tert-butyl(dimethyl)silyl]oxy}-2-fluoro-[1,1'-biphenyl]-4-carbaldehyd-
e oxime
[0089] The title compound was prepared by reacting
4'-{[tert-butyl(dimethyl)silyl]oxy}-2-fluoro-1,1'-biphenyl-4-carbaldehyde
(450 mg, 1.36 mmol) with hydroxylamine hydrochloride (190 mg, 2.73
mmol) according to Method C to yield a white solid which was used
in the next step without purification: MS (ESI) m/z 344
(M-H).sup.-, 346 (M+H).sup.+.
EXAMPLE 13
3-Fluoro-4'-methoxy-1,1'-biphenyl-4-carbaldehyde
[0090] The title compound was prepared by reacting
4-bromo-2-fluoro-benzaldehyde (3 g, 14.8 mmol) with
4-methoxyphenylboronic acid (2.70 g, 17.8 mmol) according to Method
B to yield 3.2 g (94%) of white solid: mp 85-86.degree. C.; .sup.1H
NMR (DMSO-d.sub.6): .delta. 3.83 (3H, s). 7.06-7.09 (2H, m),
7.70-7.75 (2H, m), 7.79-7.82 (2H, m), 7.88 (1H, t, J=7.96 Hz),
10.22 (1H, s); IR 1681 cm.sup.-1; MS (ESI) m/z 231 (M+H).sup.+.
[0091] Anal. for C.sub.14H.sub.11FO.sub.2:
[0092] Calc'd: C, 73.03; H, 4.82.
[0093] Found: C, 72.99; H, 4.73.
EXAMPLE 14
3-Fluoro-4'-hydroxy-1,1'-biphenyl-4-carbaldehyde
[0094] Method F. To a mixture of
3-fluoro-4'-methoxy-1,1'-biphenyl-4-carbaldehyde (0.986 g, 4.29
mmol) in methylene chloride (35 mL) at 0.degree. C. was slowly
added boron tribromide (10.7 mL of 1 N solution in methylene
chloride, 10.7 mmol). The mixture was allowed to warm slowly to
room temperature and was stirred overnight. Water (4 mL) was
injected into the mixture with stirring in an ice-water bath. The
resulting mixture was poured into water and extracted with ethyl
acetate (3.times.). The combined organic layers were washed with
brine, dried over sodium sulfate, filtered, evaporation of the
solvent and purification by silica column chromatography (15%-30%
ethyl acetate-hexane) to yield 150 mg (16%) of the title compound
as white solid. An analytical sample was afforded by reverse-phase
preparative HPLC: mp 164-166.degree. C.; .sup.1H NMR
(Acetone-d.sub.6): .delta. 76.98-7.01 (2H, m), 7.56 (1H, dd,
J=12.49 Hz, J=1.64 Hz), 7.63-7.66 (1H, m), 7.67-7.70 (2H, m), 7.89
(1H, t, J=7.85 Hz), 8.88 (1H, bs), 10.31 (1H, s); MS (ESI) m/z 215
(M-H).sup.-.
[0095] Anal. for C.sub.13H.sub.9FO.sub.2:
[0096] Calc'd: C, 72.22; H, 4.20.
[0097] Found: C, 71.83; H, 4.04.
EXAMPLE 15
Trifluoro-methanesulfonic acid 2-chloro-4-formyl-phenyl ester
[0098] The title compound was prepared by reacting
3-chloro-4-hydroxybenzaldehyde (5 g, 31.9 mmol) with
trifluoromethanesulfonic anhydride (7.0 mL, 11.7 g, 41.5 mmol)
according to Method D to yield 9.15 g (100%) of a yellow oil which
was used directly in the next step without purification: .sup.1H
NMR: .delta. 7.92 (1H, d, J=8.48 Hz), 8.07 (1H, dd, J=8.51 Hz,
J=1.93 Hz), 8.30 (1H, d, J=1.92 Hz), 10.04 (1H, s).
EXAMPLE 16
2-Chloro-4'-methoxy-1,1'-biphenyl-4-carbaldehyde
[0099] The title compound was prepared by reacting
trifluoro-methanesulfonic acid 2-chloro-4-formyl-phenyl ester (5 g,
17.4 mmol) with 4-methoxyphenylboronic acid (3.44 g, 22.6 mmol)
according to Method B to yield 3.83 g (89%) of white solid: mp
85-87.degree. C.; .sup.1H NMR (DMSO-d.sub.6): .delta. 3.82 (3H, s),
7.07 (2H, d, J=8.83 Hz), 7.46 (2H, d, J=8.45 Hz), 7.63 (1H, d,
J=7.87 Hz), 7.92 (1H, dd, J=7.87 Hz, J=1.56 Hz), 8.07 (1H, d,
J=1.49 Hz); .sup.13C NMR (DMSO-d.sub.6): .delta. 55.14, 113.72,
127.80, 129.72, 130.42, 130.82, 132.15, 132.21, 136.11, 144.90,
159.33, 191.73; IR 1692 cm.sup.-1; MS (EI) m/z 246/248 M.sup.+.
[0100] Anal. for C.sub.14H.sub.11ClO.sub.2:
[0101] Calc'd: C, 68.16; H, 4.49.
[0102] Found: C, 67.76; H, 4.36.
EXAMPLE 17
2'-Chloro-4'-(dibromomethyl)-1,1'-biphenyl-4-ol
[0103] The title compound was prepared by reacting
2-chloro-4'-methoxy-1,1'-biphenyl-4-carbaldehyde (800 mg, 3.25
mmol) with boron tribromide (8.1 mL of 1 N solution in methylene
chloride, 8.13 mmol) according to Method F to yield 600 mg (50%) of
yellowish solid: mp 107-108.degree. C.; .sup.1H NMR (DMSO-d.sub.6):
.delta. 6.86 (2H, d, J=8.52 Hz), 7.29 (2H, d, J=8.50 Hz), 7.43 (1H,
s), 7.45 (1H, d, J=8.12 Hz), 7.64 (1H, dd, J=8.07 Hz, J=1.83 Hz),
7.74 (1H, d, J=1.79 Hz), 9.71 (1H, bs); .sup.13C NMR
(DMSO-d.sub.6): .delta. 40.66, 114.94, 125.66, 127.34, 128.14,
130.39, 130.87, 131.80, 140.95, 142.14, 157.36; MS (ESI) m/z
373/375/377/379 (M-H).sup.-.
[0104] Anal. for C.sub.13H.sub.9Br.sub.2ClO:
[0105] Calc'd: C, 41.48; H, 2.41.
[0106] Found: C, 41.64; H, 2.14.
EXAMPLE 18
Trifluoro-methanesulfonic acid 4-formyl-3-methoxy-phenyl ester
[0107] The title compound was prepared by reacting
4-hydroxy-2-methoxybenzaldehyde (3 g, 19.7 mmol) with
trifluoromethanesulfonic anhydride (4.3 mL, 7.2 g, 25.6 mmol)
according to Method D to yield a brown syrup which was used in the
next step without purification: .sup.1H NMR: .delta. 3.97 (3H, s),
7.21 (1H, dd, J=8.64 Hz, J=2.17 Hz), 7.47 (1H, d, J=2.24 Hz), 7.87
(1H, d, J=8.64 Hz), 10.31 (1H, s).
EXAMPLE 19
Trifluoro-methanesulfonic acid 4-formyl-2-methyl-phenyl ester
[0108] The title compound was prepared by reacting
4-hydroxy-3-methylbenzaldehyde (2.5 g, 18.4 mmol) with
trifluoromethanesulfonic anhydride (4.0 mL, 6.75 g, 23.9 mmol)
according to Method D to yield a brown oil which was used directly
in the next step without purification.
EXAMPLE 20
4'-Hydroxy-3-methoxy-1,1'-biphenyl-4-carbaldehyde
[0109] The title compound was prepared by reacting
trifluoro-methanesulfonic acid 4-formyl-3-methoxy-phenyl ester
(11.0 mmol) with 4-tert-butyl-dimethylsilyoxyphenyl-boronic acid
(3.50 g, 13.86 mmol) according to Method B to yield 880 mg (35%,
over two steps) of yellowish solid: mp 159-161.degree. C.; .sup.1H
NMR (DMDO-d.sub.6): .delta. 4.01 (3H, s), 6.89 (2H, d, J=8.62 Hz),
7.31 (1H, d, J=8.16 Hz), 7.36 (1H, d, J=1.15 Hz), 7.66 (2H, d,
J=8.63 Hz), 7.72 (1H, d, J=8.09 Hz), 9.80 (1H, s), 10.34 (1H, s);
mp 159-161.degree. C.; IR 1660 cm.sup.-1; MS (ESI) m/z 227
(M-H).sup.-, 229 (M+H).sup.+.
[0110] Anal. for C.sub.14H.sub.12O.sub.3:
[0111] Calc'd: C, 73.67; H, 5.30.
[0112] Found: C, 73.44; H, 4.99.
EXAMPLE 21
4'-Hydroxy-2-methyl-1,1'-biphenyl-4-carbaldehyde
[0113] The title compound was prepared by reacting
trifluoro-methanesulfonic acid 4-formyl-2-methyl-phenyl ester (9.3
mmol) with 4-tert-butyl-dimethylsilyoxyphenylboronic acid (2.35 g,
9.3 mmol) according to Method B to yield 1.12 mg (57%, over two
steps) of yellowish solid: mp 94-96.degree. C.; .sup.1H NMR
(DMSO-d.sub.6): .delta. 2.33 (3H, s), 6.86 (2H, d, J=8.55 Hz), 7.22
(2H, d, J=8.51 Hz), 7.39 (1H, d, J=7.81 Hz), 7.61 (1H, d, J=7.82
Hz), 7.81 (1H, s), 9.65 (1H, s), 10.00 (1H, s); IR 1670 cm.sup.-1;
MS (ESI) m/z 211 (M-H).sup.-, 213 (M+H).sup.+.
[0114] Anal. for C.sub.14H.sub.12O.sub.2:
[0115] Calc'd: C, 79.23; H, 5.70.
[0116] Found: C, 77.49; H, 5.67.
EXAMPLE 22
3-Fluoro-4-methoxyphenylboronic acid
[0117] The title compound was prepared by reacting
4-bromo-2-fluoroanisole (10 g, 0.049 moles) with n-butyl lithium
(23.4 mL of 2.5 M solution in hexane, 0.059 moles) followed by
triisopropyl borate (45.2 mL, 36.9 g, 0.196 moles) according to
Method A to yield 7.1 g (85.2%) of a white solid: MS (ESI) m/z 169
(M-H).sup.-.
EXAMPLE 23
3-Chloro-3'-fluoro-4'-methoxy-1,1'-biphenyl-4-carbaldehyde
[0118] The title compound was prepared by reacting
trifluoro-methanesulfonic acid 3-chloro-4-formyl-phenyl ester (2.8
g, 9.62 mmol) with 3-fluoro-4-methoxyphenylboronic acid (1.8 g,
10.6 mmol) according to Method B to yield 1.87 g (74%, over two
steps) of white solid: mp 116-118.degree. C.; .sup.1H NMR
(DMSO-d.sub.6): .delta. 3.91 (3H, s), 7.30 (1H, t, J=8.79 Hz),
7.66-7.68 (1H, m), 7.79 (1H, dd, J=12.91 Hz, J=2.47 Hz), 7.85-7.87
(1H, m), 7.91 (1H, d, J=8.24 Hz), 7.96 (1H, d, J=1.65 Hz), 10.34
(1H, s); IR 1688 cm.sup.-1; MS (ESI) m/z 265/267 (M+H).sup.+.
[0119] Anal. for C.sub.14H.sub.10ClFO.sub.2:
[0120] Calc'd: C, 63.53; H, 3.81.
[0121] Found: C, 63.29; H, 3.63.
EXAMPLE 24
3-Chloro-3'-fluoro-4'-hydroxy-1,1'-biphenyl-4-carbaldehyde
[0122] The title compound was prepared by reacting
3-chloro-3'-fluoro-4'-methoxy-1,1'-biphenyl-4-carbaldehyde (990 mg,
3.75 mmol) with boron tribromide (11.25 mL of 1 N solution in
methylene chloride, 11.25 mmol) according to Method F to yield 940
mg (100%) of yellowish solid. The compound was used in the next
step without purification. The analytical sample was obtained by
HPLC purification: mp 206-208.degree. C.; .sup.1H NMR
(DMSO-d.sub.6): .delta. 7.06 (1H, t, J=8.79 Hz), 7.51-7.53 (1H, m),
7.71 (1H, dd, J=2.47 Hz, J=12.63 Hz), 7.81-7.83 (1H, m), 7.89 (1H,
d, J=7.96 Hz), 7.91 (1H, d, J=1.66 Hz), 10.329 (1H, s), 10.330 (1H,
s); IR 1667 cm.sup.-1; MS (ESI) m/z 249/251 (M-H).sup.-.
[0123] Anal. for C.sub.13H.sub.8ClFO.sub.2:
[0124] Calc'd: C, 62.29; H, 3.22.
[0125] Found: C, 61.97; H, 3.21.
EXAMPLE 25
3-Chloro-3',5'-difluoro-4'-hydroxy-1,1'-biphenyl-4-carbaldehyde
[0126] The title compound was prepared by reacting
trifluoro-methanesulfonic acid 3-chloro-4-formyl-phenyl ester (1 g,
3.5 mmol) with 3,5-difluoro-4-tert-butyldimethylsilyoxy-boronic
acid (1.1 g, 3.8 mmol) according to Method B to yield 0.56 g (60%)
of yellow solid: mp 233-235.degree. C.; .sup.1H NMR (DMSO-d.sub.6):
.delta. 7.64 (2H, d, J=7.85 Hz), 7.85-7.91 (2H, m), 7.98 (1H, s),
10.33 (1H, s), 10.70 (1H, bs); IR 1665 cm.sup.-1; MS (ESI) m/z
(267/269) (M-H).sup.-.
[0127] Anal. for C.sub.13H.sub.7ClF.sub.2O.sub.2:
[0128] Calc'd: C, 58.12; H, 2.63.
[0129] Found: C, 57.79; H, 2.72.
EXAMPLES 26 AND 27
[0130] Trifluoro-methanesulfonic acid 3-chloro-4-formyl-phenyl
ester (3.78 g, 13.12 mmol) was reacted with
4-methoxy-2-methylphenylboronic acid (2.61 g, 15.7 mmol) according
to Method B to produce two compounds:
3-Chloro-4'-methoxy-2'-methyl-1,1'-biphenyl-4-carbaldehyde
[0131] 2.18 g (64%) white solid: mp 77-79.degree. C.; .sup.1H NMR
(DMSO-d.sub.6): .delta. 2.26 (3H, s), 3.79 (3H, s), 6.88 (1H, dd,
J=8.41 Hz, J=2.66 Hz), 6.93 (1H, d, J=2.50 Hz), 7.23 (1H, J=8.40
Hz), 7.50 (1H, dd, J=7.93 Hz, J=1.32 Hz), 7.58 (1H, d, J=1.53 Hz),
7.91 (1H, d, J=7.57 Hz), 10.36 (1H, s); IR 1682 cm.sup.-1; MS (ESI)
m/z 261/263 (M+H).sup.+.
[0132] Anal. for C.sub.15H.sub.13ClO.sub.2:
[0133] Calc'd: C, 69.10; H, 5.03.
[0134] Found: C, 69.13; H, 4.73.
4,4''-Dimethoxy-2,2''-dimethyl-1,1':3',1''-terphenyl-4'-carbaldehyde
[0135] 0.56 g (12%) colorless; .sup.1H NMR (DMSO-d.sub.6): .delta.
2.10 (3H, s), 2.29 (3H, s), 3.78 (3H, s), 3.80 (3H, s), 6.85-6.88
(2H, m), 6.91 (1H, d, J=2.38 Hz), 6.95 (1H, d, J=2.38 Hz), 7.18
(1H, d, J=8.32 Hz), 7.24-7.25 (2H, m), 7.52 (1H, dd, J=7.74 Hz,
J=1.78 Hz), 7.95 (1H, d, J=8.33 Hz), 9.68 (1H, s); IR 1679
cm.sup.-1; MS (ESI) m/z 347 (M+H).sup.+.
[0136] Anal. for C.sub.23H.sub.22O.sub.3:
[0137] Calc'd: C, 79.74; H, 6.40.
[0138] Found: C, 79.25; H, 6.05.
EXAMPLE 28
3-Chloro-4'-hydroxy-2'-methyl-1,1'-biphenyl-4-carbaldehyde
[0139] 3-Chloro-4'-methoxy-2'-methyl-1,1'-biphenyl-4-carbaldehyde
(1.0 g, 3.84 mmol) and pyridinium HCl (6 g) in a sealed tube was
heated at 195.degree. C. with stirring for 1 hr. The reaction
mixture was cooled to room temperature and stirred with 2 N HCl
solution and ethyl acetate. The organic layer was separated and the
aqueous layer was extracted with ethyl acetate (.times.2). The
combined organic layers were washed with brine, dried over sodium
sulfate, filtered, and evaporation of the solvent provided (1 g)
dark green solid. The product was used without any further
purification. An analytical sample was obtained by HPLC
purification giving a white solid; mp 125-127.degree. C.; .sup.1H
NMR (DMSO-d.sub.6): .delta. 2.21 (3H, s), 6.68-6.72 (2H, m), 7.11
(1H, d, J=8.15 Hz), 7.47 (1H, d, J=7.89 Hz), 7.55 (1H, d, J=1.48
Hz), 7.89 (1H, d, J=7.98 Hz), 9.62 (1H, s), 10.2=35 (1H, s); MS
(ESI) m/z 245/247 (M-H).sup.-, 247/249 (M+H).sup.+.
[0140] Anal. for C.sub.14H.sub.11ClO.sub.2:
[0141] Calc'd: C, 68.16; H, 4.49.
[0142] Found: C, 67.63; H, 4.43.
EXAMPLE 29
4'-Hydroxy-3-methyl[1,1'-biphenyl]-4-carbaldehyde oxime
[0143] A mixture of
4'-hydroxy-3-methyl[1,1'-biphenyl]-4-carbaldehyde (310 mg, 1.46
mmol), hydroxylamine hydrochloride (203 mg, 2.92 mmol) and pyridine
(236 ul, 2.92 mmol) in 15 mL absolute methanol was refluxed for 2.5
h. The solvent was removed under reduced pressure and the mixture
was dissolved in ethyl acetate and water, extracted with ethyl
acetate (.times.3), washed with brine, dried over sodium sulfate,
and filtered. Evaporation of the solvent and purification by
recrystallization (ethyl acetate, acetone and hexane) gave 177 mg
(53%) of the title compound as a yellowish solid: mp
195-197.degree. C.;
[0144] .sup.1H NMR (DMSO-d.sub.6): .delta. 2.43 (3H, s), 6.84 (2H,
d, J=8.48 Hz), 7.42-7.45 (2H, m), 7.52 (2H, d, J=8.51 Hz), 7.66
(1H, d, J=8.00 Hz), 8.32 (1H, s), 9.60 (1H, s), 11.25 (1H, s);
.sup.13C NMR (DMSO-d.sub.6): .delta. 19.62, 115.60, 123.40, 126.60,
127.56, 127.99, 129.05, 130.03, 136.34, 140.43, 146.80, 157.24; MS
(ESI) m/z 226 (M-H).sup.-, 228 (M+H).sup.+.
[0145] Anal. for C.sub.14H.sub.13NO.sub.2:
[0146] Calc'd: C, 73.99; H, 5.77; N, 6.16.
[0147] Found: C, 73.81; H, 5.75; N, 6.04.
EXAMPLE 30
4'-Hydroxy[1,1'-biphenyl]-4-carbaldehyde oxime
[0148] The title compound was prepared by reacting
4'-hydroxy[1,1'-biphenyl]-4-carbaldehyde (1.03 mmol) with
hydroxylamine hydrochloride (140 mg, 2 mmol) according to Method C
to yield 193 mg (79%, over two steps) of yellowish solid: mp
207-210.degree. C.; .sup.1H NMR (DMSO-d.sub.6): .delta. 6.85 (2H,
d, J=8.37 Hz), 7.53 (2H, d, J=8.39 Hz), 7.62 (4H, s), 8.15 (1H, s),
9.62 (1H, s), 11.21 (1H, s); MS (ESI) m/z 212 (M-H).sup.-.
[0149] Anal. for C.sub.13H.sub.11NO.sub.2:
[0150] Calc'd: C, 73.23; H, 5.20; N, 6.57.
[0151] Found: C, 72.78; H, 5.41; N, 6.38.
EXAMPLE 31
3-Chloro-4'-hydroxy[1,1'-biphenyl]-4-carbaldehyde oxime
[0152] Method E. Tetrabutylammonium fluoride (1.29 mL of 1.0 M
solution in tetrahydrofuran, 1.29 mmol) was added into a solution
of
4'-{[tert-butyl(dimethyl)silyl]oxy}-3-chloro[1,1'-biphenyl]-4-carbaldehyd-
e oxime (1.17 mmol) in 10 mL tetrahydrofuran. The mixture was
stirred at room temperature for 10 min then poured into ethyl
acetate and water. The resulting layers were separated and the
aqueous layer was extracted with ethyl acetate (3.times.). The
combined organic layers were washed with brine, dried over sodium
sulfate, filtered, and the solvent removed under vacuum. Purified
by silica chromatography (20%-30% ethyl acetate-hexane) provided
0.186 mg (64%) of the title compound as yellowish solid: mp
187-189.degree. C.; .sup.1H NMR (DMSO-d.sub.6): .delta. 6.86 (2H,
d, J=8.55 Hz), 7.56-7.63 (3H, m), 7.71 (1H, d, J=1.60 Hz), 7.84
(1H, d, J=8.26 Hz), 8.36 (1H, s), 9.75 (1H, s), 11.66 (1H, s); MS
(ESI) m/z 246 (M-H).sup.-, 248 (M+H).sup.+.
[0153] Anal. for C.sub.13H.sub.10ClNO.sub.2:
[0154] Calc'd: C, 63.04; H, 4.07; N, 5.66.
[0155] Found: C, 62.96; H, 4.10; N, 5.42.
EXAMPLE 32
2-Fluoro-4'-hydroxy-1,1'-biphenyl-4-carbaldehyde oxime
[0156] The title compound was prepared by reacting
4'-{[tert-butyl(dimethyl)silyl]oxy}-2-fluoro-[1,1'-biphenyl]-4-carbaldehy-
de oxime (1.02 mmol) with tetrabutylammonium fluoride (1.12 mL of
1.0 M solution in tetrahydrofuran, 1.12 mmol) according to Method E
to yield 198 mg (84%) of a white solid: mp 178-180.degree. C.;
.sup.1H NMR (DMSO-d.sub.6): .delta. 6.84-6.89 (2H, m), 7.39-5.54
(5H, m), 8.17 (1H, s), 9.71 (1H, s), 11.43 (1H, s); MS (ESI) m/z
230 (M-H).sup.-, 232 (M+H).sup.+.
[0157] Anal. for C.sub.13H.sub.10FNO.sub.2:
[0158] Calc'd: C, 67.53; H, 4.36; N, 6.06.
[0159] Found: C, 67.13; H, 4.11; N, 6.00.
EXAMPLE 33
3-Fluoro-4'-hydroxy-1,1'-biphenyl-4-carbaldehyde oxime
[0160] The title compound was prepared by reacting
3-fluoro-4'-hydroxy-1,1'-biphenyl-4-carbaldehyde (154 mg, 0.713
mmol) with hydroxylamine hydrochloride (99 mg, 1.43 mmol) according
to Method C to yield 156 mg (95%) of yellowish solid: mp
181-183.degree. C.; .sup.1H NMR (DMSO-d.sub.6): .delta. 6.84-6.87
(2H, m), 7.48-7.53 (2H, m), 7.57-7.60 (2H, m), 7.76 (1H, t, J=8.14
Hz), 8.22 (1H, s), 9.74 (1H, s), 11.56 (1H, s); MS (ESI) m/z 230
(M-H).sup.-, 232 (M+H).sup.+.
[0161] Anal. for C.sub.13H.sub.10FNO.sub.2:
[0162] Calc'd: C, 67.53; H, 4.36; N, 6.06.
[0163] Found: C, 68.10; H, 4.28; N, 6.01.
EXAMPLE 34
2-Chloro-4'-hydroxy-1,1'-biphenyl-4-carbaldehyde oxime
[0164] The title compound was prepared by reacting
2'-chloro-4'-(dibromomethyl)-1,1'-biphenyl-4-ol (270 mg, 0.722
mmol) with hydroxylamine hydrochloride (185 mg, 2.66 mmol)
according to Method C to yield 160 mg (90%) of white solid: mp
178-179.degree. C.; .sup.1H NMR (DMSO-d.sub.6): .delta. 6.85 (2H,
d, J=8.54 Hz), 7.28 (2H, d, J=8.51 Hz), 7.39 (1H, d, J=7.98 Hz),
7.60 (1H, dd, J=7.65 Hz, J=1.42 Hz), 7.72 (1H, d, J=1.34 Hz), 8.18
(1H, s), 9.67 (1H, s), 11.45 (1H, s); MS (ESI) m/z 246/248
(M-H).sup.-.
[0165] Anal. for C.sub.13H.sub.10ClNO.sub.2:
[0166] Calc'd: C, 63.04; H, 4.07; N, 5.66.
[0167] Found: C, 63.07; H, 3.99; N, 5.67.
EXAMPLE 35
4'-Hydroxy-3-methoxy-1,1'-biphenyl-4-carbaldehyde oxime
[0168] The title compound was prepared by reacting
4'-hydroxy-3-methoxy-1,1'-biphenyl-4-carbaldehyde (225 mg, 0.986
mmol) with hydroxylamine hydrochloride (137 mg, 1.97 mmol)
according to Method C to yield 210 mg (88%) of yellowish solid: mp
228-230.degree. C.; .sup.1H NMR (DMSO-d.sub.6): .delta. 3.91 (3H,
s), 6.85 (2H, d, J=8.29 Hz), 7.18 (1H, d, J=8.08 Hz), 7.21 (1H, s),
7.56 (2H, d, J=8.59 Hz), 7.68 (1H, d, J=7.98 Hz), 8.28 (1H, s),
9.63 (1H, s), 11.19 (1H, s); MS (ESI) m/z 242 (M-H).sup.-, 244
(M+H).sup.+.
[0169] Anal. for C.sub.14H.sub.13NO.sub.3:
[0170] Calc'd: C, 69.12; H, 5.39; N, 5.76.
[0171] Found: C, 68.87; H, 5.29; N, 5.64.
EXAMPLE 36
4'-Hydroxy-2-methyl-1,1'-biphenyl-4-carbaldehyde oxime
[0172] The title compound was prepared by reacting
4'-hydroxy-2-methyl-1,1'-biphenyl-4-carbaldehyde (350 mg, 1.65
mmol) with hydroxylamine hydrochloride (230 mg, 3.30 mmol)
according to Method C to yield 360 mg (96%) of white solid: mp
169-171.degree. C.;
[0173] .sup.1H NMR (DMSO-d.sub.6): .delta. 2.25 (3H, s), 6.82 (2H,
d, J=8.52 Hz), 7.16 (2H, d, J=8.45 Hz), 7.18 (1H, d, J=7.84 Hz),
7.44 (1H, d, J=7.93 Hz), 7.47 (1H, s), 8.11 (1H, s), 9.52 (1H, s),
11.19 (1H, s); MS (ESI) m/z 226 (M-H).sup.-, 228 (M+H).sup.+.
[0174] Anal. for C.sub.14H.sub.13NO.sub.2:
[0175] Calc'd: C, 73.99; H, 5.77; N, 6.16.
[0176] Found: C, 73.72; H, 5.63; N, 6.37.
EXAMPLE 37
3-Chloro-3'-fluoro-4'-hydroxy-1,1'-biphenyl-4-carbaldehyde
oxime
[0177] The title compound was prepared by reacting
3-chloro-3'-fluoro-4'-hydroxy-1,1'-biphenyl-4-carbaldehyde (1.52
mmol) with hydroxylamine hydrochloride (233 mg, 3.34 mmol)
according to Method C to yield 260 mg (66%) of yellowish solid: mp
198-200.degree. C.; .sup.1H NMR (DMSO-d.sub.6): .delta. 7.03 (1H,
t, J=8.79 Hz), 7.41-7.43 (1H, m), 7.60 (1H, dd, J=12.91 Hz, J=2.20
Hz), 7.64-7.66 (1H, m), 7.77 (1H, d, J=1.92 Hz), 7.84 (1H, d,
J=8.24 Hz), 8.36 (1H, s), 10.16 (1H, bs), 11.68 (1H, bs); MS (ESI)
m/z 264/266 (M-H)--, 266/268 (M+H).sup.+.
[0178] Anal. for C.sub.13H.sub.9ClFNO.sub.2:
[0179] Calc'd: C, 58.77; H, 3.41; N, 5.27.
[0180] Found: C, 58.67; H, 3.65; N, 4.99.
EXAMPLE 38
3-Chloro-3',5'-fluoro-4'-hydroxy-1,1'-biphenyl-4-carbaldehyde
oxime
[0181] The title compound was prepared by reacting
3-chloro-3',5'-difluoro-4'-hydroxy-1,1'-biphenyl-4-carbaldehyde
(200 mg, 0.746 mmol) with hydroxylamine hydrochloride (156 mg, 2.24
mmol) according to Method C to yield 100 mg (47%) of white
solid:
[0182] mp 216-219.degree. C.; .sup.1H NMR (DMSO-d.sub.6): .delta.
7.54 (2H, d, J=8.51 Hz), 7.70 (1H, d, J=7.97 Hz), 7.84-7.86 (2H,
m), 8.37 (1H, s), 10.52 (1H, s), 11.74 (1H, s); MS (ESI) m/z
282/284 (M-H).sup.-, 284/286 (M+H).sup.+.
[0183] Anal. for C.sub.13H.sub.8ClF.sub.2NO.sub.2:
[0184] Calc'd: C, 55.05; H, 2.84; N, 4.94.
[0185] Found: C, 54.96; H, 3.02; N, 4.75.
EXAMPLE 39
3-Chloro-4'-hydroxy-2'-methyl-1,1'-biphenyl-4-carbaldehyde
oxime
[0186] The title compound was prepared by reacting
3-chloro-4'-hydroxy-2'-methyl-1,1'-biphenyl-4-carbaldehyde (500 mg,
2.03 mmol) with hydroxylamine hydrochloride (425 mg, 6.10 mmol) in
anhydrous tetrahydrofuran (50 mL) and methanol (20 mL) according to
Method C to yield 350 mg (57% over two steps) of white solid: mp
169-171.degree. C.; .sup.1H NMR (DMSO-d.sub.6): .delta. 2.19 (3H,
s), 6.65-6.70 (2H, m), 7.06 (1H, d, J=8.14 Hz), 7.31 (1H, d, J=8.05
Hz), 7.41 (1H, d, J=1.40 Hz), 7.83 (1H, d, J=8.08 Hz), 8.38 (1H,
s), 9.51 (1H, s), 11.69 (1H, s); MS (ESI) m/z 260/262 (M-H).sup.-,
262/264 (M+H).sup.+.
[0187] Anal. calcd for C.sub.14H.sub.12ClNO.sub.2:
[0188] Calc'd: C, 64.25; H, 4.62; N, 5.35.
[0189] Found: C, 63.87; H, 4.43; N, 5.31.
EXAMPLE 40
(2,6-Dichloro-4-methoxy-phenyl)-methanol
[0190] A mixture of 3,5 dichloroanisole (16.39 g, 92.59 mmol), HCl
(250 mL of concentrated solution), and sulfuric acid (2.5 mL of
concentrated solution) was stirred at 60.degree. C. overnight. The
mixture was cooled to room temperature and the organic layer was
removed. The aqueous layer was extracted with dichloromethane
(2.times.100 mL). The combined organic layers were washed with
water and the solvent was removed by evaporation. To the remaining
oil was added NaOH (180 mL of 1N solution) and dioxane (85 mL). The
mixture was stirred at reflux for 3 hours and cooled to room
temperature. The organic layer was removed and the aqueous layer
was extracted with dichloromethane (3.times.100 mL). The combined
organic layers were washed with water, washed with brine, dried
over sodium sulfate, and filtered. The residue was purified on
silica (90% hexanes-10% ethyl acetate) to yield 5.82 g (30%) of the
title compound as a white solid: mp 71-72.degree. C.; .sup.1H NMR
(CDCl.sub.3): .delta. 1.88 (1H, s), 3.73 (3H, s), 4.81 (2H, s),
6.81 (2H, s); MS (EI) m/z 206/208/210 (M.sup.+).
[0191] Anal. for C.sub.8H.sub.8Cl.sub.2O.sub.2:
[0192] Calc'd: C, 46.41; H, 3.89.
[0193] Found: C, 46.38; H, 3.69.
EXAMPLE 41
2,6-Dichloro-4-methoxy-benzaldehyde
[0194] A suspension of (2,6-dichloro-4-methoxy-phenyl)-methanol
(5.69 g, 27.49 mmol) and MnO.sub.2 (15 g) in benzene (100 mL) was
stirred at reflux, utilizing a Dean-Stark trap, overnight. The
suspension was cooled to room temperature, filtered through Celite,
and the solvent was removed by evaporation to yield 4.69 g (83%) of
crude white solid. An analytical sample was obtained by
recrystallization from methanol to yield white needle crystals: mp
104-106.degree. C.; .sup.1H NMR (DMSO-d.sub.6): .delta. 3.90 (3H,
s), 7.21 (2H, s), 10.29 (1H, s); MS (EI) m/z 204/206/218
(M.sup.+).
[0195] Anal. for C.sub.8H.sub.6Cl.sub.2O.sub.2:
[0196] Calc'd: C, 46.86; H, 2.
[0197] Found: C, 46.67; H, 2.89.
EXAMPLE 42
2,6-Dichloro-4-hydroxy-benzaldehyde
[0198] To a solution of 2,6-dichloro-4-methoxy-benzaldehyde (3.44
g, 16.8 mmol) in dichloromethane (120 mL) at 0.degree. C. was
slowly added boron tribromide (42 mL of 1N in dichloromethane, 42
mmol). The solution was allowed to warm to room temperature while
stirring overnight and was quenched with saturated sodium
bicarbonate solution (250 mL). The resulting mixture was extracted
with ethyl acetate (3.times.200 mL). The combined organic layers
were washed with water, washed with brine, dried over sodium
sulfate, and filtered. The solvent was removed by evaporation and
the residue was purified on silica (70% hexanes-30% ethyl acetate)
to yield 2.19 g (68%) of a pink solid. Trituration with ethyl
acetate--hexanes yielded an analytical sample of the title compound
as a white solid: mp: 214-217.degree. C.; .sup.1H NMR
(DMSO-d.sub.6): .delta. 6.95 (2H, s), 10.26 (1H, s), 11.44 (1H, s);
MS (EI) m/z 189.8/191.8/193.8 (M.sup.+).
[0199] Anal. for C.sub.7H.sub.4Cl.sub.2O.sub.2:
[0200] Calc'd: C, 44.02; H, 2.11.
[0201] Found: C, 44.08; H, 2.07.
EXAMPLE 43
3,5-Dichloro-4-formyl-phenyl trifluoromethanesulfonate
[0202] The title compound was prepared by reacting
2,6-dichloro-4-hydroxy-benzaldehyde (2.35 g, 12.3 mmol) with
trifluoromethanesulfonic anhydride (4.51 g, 16.0 mmol) according to
method E to yield a 3.45 g (87%) of a clear yellow oil. TLC
analysis of this oil indicated that it appeared to decompose into
the starting phenol upon standing. .sup.1H NMR (DMSO-d.sub.6):
.delta. 8.03 (2H, s), 10.31 (1H, s).
EXAMPLE 44
3,5-Dichloro-4'hydroxy-biphenyl-4-carbaldehyde
[0203] The title compound was prepared by reacting
3,5-dichloro-4-formyl-phenyl trifluoromethanesulfonate (0.73 g,
2.26 mmol) with 4-tert-butyldimethylsilyloxyphenyl boronic acid
(0.80 g, 3.2 mmol) according to method C to yield 0.30 g (50%) of a
yellow solid: mp: 178-180.degree. C.; .sup.1H NMR (DMSO-d.sub.6):
.delta. 6.88 (2H, d, J=8.84 Hz), 7.72 (2H, d, J=8.71 Hz), 7.84 (2H,
s), 9.95 (1H, s), 10.38 (1H, s); MS (EI) m/z 266.0/268.0/270.0
(M.sup.+).
[0204] Anal. for C.sub.13H.sub.8Cl.sub.2O.sub.2. 0.5H.sub.2O:
[0205] Calc'd: C, 56.55; H, 3.29.
[0206] Found: C, 56.36; H, 2.90.
EXAMPLE 45
2,3-Dichloro-4-methoxybenzaldehyde
[0207] To a solution of 2,3-dichloroanisole (10.00 g, 56.6 mmol) in
anhydrous dichloromethane (45 ml) was added TiCl.sub.4 (10.5 ml,
96.1 mmol) quickly. .alpha., .alpha.'-dichloromethyl methyl ether
(5.1 ml, 56.6 mmol) was then added slowly and the inner temperature
was maintained between 15.degree. C. to 20.degree. C. The mixture
was stirred room temperature for 5 h, poured into crushed ice
slowly, extracted with dichloromethane (3.times.), washed with
[0208] saturated sodium bicarbonate until pH=7, then washed with
brine. The organic layer was dried over anhydrous sodium sulfate,
concentrated to give 11.34 g (98%) of white solid. An analytical
sample was afforded by reverse-phase preparative HPLC: mp
112-113.degree. C.; .sup.1H NMR (CDCl.sub.3): .delta. 4.01 (3H, s),
6.97 (1H, d, J=8.77 Hz), 7.90 (1H, d, J=8.82 Hz), 10.36 (1H, s); MS
(ESI) m/z 205/207/209 (M+H).sup.+.
[0209] Anal. for C.sub.8H.sub.6Cl.sub.2O.sub.2:
[0210] Calc'd: C, 46.86; H, 2.95.
[0211] Found: C, 46.92; H, 2.70.
EXAMPLE 46
2,3-Dichloro-4-hydroxybenzaldehyde
[0212] The title compound was prepared by reacting
2,3-dichloro-4-methoxybenzaldehyde (10 g, 49 mmol) with boron
tribromide (147 ml of 1N solution in CH.sub.2Cl.sub.2, 147 mmol)
according to method D to yield 11.3 g dark grey solid which is
mainly the designed product indicated by .sup.1H-NMR. It was
triturated with 30% CHCl.sub.3 in hexane to give 3.6 g of grey
solid as pure product. An analytical sample was afforded as white
solid by reverse-phase preparative HPLC: mp 176-178.degree. C.;
.sup.1H NMR (DMSO-d.sub.6): .delta. 7.10 (1H, d, J=8.68 Hz), 7.74
(1H, d, J=8.67 Hz), 10.16 (1H, s), 11.95 (1H, s); MS (ESI) m/z
189/191/193 (M-H).sup.-, 193/191/195 (M+H).sup.+.
[0213] Anal. for C.sub.7H.sub.4Cl.sub.2O.sub.2
[0214] Calc'd: C, 44.02; H, 2.11.
[0215] Found: C, 43.87; H, 1.67.
EXAMPLE 47
Trifluoro-methanesulfonic acid 2,3-dichloro-4-formyl-phenyl
ester
[0216] The title compound was prepared by reacting
2,3-dichloro-4-hydroxybenzaldehyde (2.50 g, 13.2 mmol) with
trifluoromethanesulfonic anhydride (2.88 ml, 4.80 g, 17.1 mmol)
according to example 43 to yield 3.69 g (82%) of a brown crystal
which was used directly in the next step without purification. An
analytical sample was afforded as white solid by silica
chromatography (5% ethyl acetate-hexane): mp 44-45.degree. C.;
.sup.1H NMR (DMSO-d.sub.6): .delta. 7.88 (1H, d, J=8.74 Hz), 8.02
(1H, d, J=8.81 Hz), 10.28 (1H, s); MS (EI) m/z 322/324/326
(M).sup.+.
[0217] Anal. for C.sub.8H.sub.3Cl.sub.2F.sub.3O.sub.4S:
[0218] Calc'd: C, 29.74; H, 0.94.
[0219] Found: C, 30.19; H, 0.86.
EXAMPLES 48-50
[0220] Trifluoro-methanesulfonic acid 2,3-dichloro-4-formyl-phenyl
ester (1.39 g, 4.33 mmol) was reacted with boronic acid 21 (1.20 g,
4.76 mmol) according to method B to produce the following three
compounds:
2,3-Dichloro-4'-hydroxy-1,1'-biphenyl-4-carbaldehyde (48)
[0221] 310 mg (27%) of white solid: mp 172-174.degree. C.; .sup.1H
NMR (DMSO-d.sub.6): .delta. 6.86-6.91 (2H, m), 7.32-7.35 (2H, m),
7.53 (1H, dd, J=7.94 Hz, J=0.45 Hz), 7.85 (1H, d, J=8.01 Hz), 9.85
(1H, s), 10.35 (1H, s); MS (ESI) m/z 265/267/269 (M-H).sup.-.
[0222] Anal. for C.sub.13H.sub.8Cl.sub.2O.sub.2:
[0223] Calc'd: C, 58.46; H, 3.02.
[0224] Found: C, 57.75; H, 2.82.
2-Chloro-4'-hydroxy-1,1'-biphenyl-4-carbaldehyde (49)
[0225] 90 mg (9%) of off white solid: mp 114-116.degree. C.;
.sup.1H NMR (DMSO-d.sub.6): .delta. 6.85-6.90 (2H, m), 7.32-7.36
(2H, m), 7.61 (1H, d, J=7.87 Hz), 7.89 (1H, dd, J=7.90 Hz, J=1.55
Hz), 8.05 (1H, d, J=1.52 Hz), 9.79 (1H, s), 10.02 (1H, s); MS (ESI)
m/z 231/233 (M-H).sup.-, 233/235 (M+H).sup.+.
[0226] Anal. for C.sub.13H.sub.9ClO.sub.2:
[0227] Calc'd: C, 67.11; H, 3.90.
[0228] Found: C, 67.44; H, 3.87.
2'-Dichloro-4,4''-dihydroxy-1,1':3',1''-terphenyl-4'-carbaldehyde
(50)
[0229] 130 mg (9%) of off white solid: mp 222-223.degree. C.;
.sup.1H NMR (DMSO-d.sub.6): .delta. 6.85-6.88 (2H, m), 6.88-6.91
(2H, m), 7.17-7.20 (2H, m), 7.31-7.35 (2H, m), 7.52 (1H, dd, J=7.96
Hz, J=0.89 Hz), 7.84 (1H, d, J=8.07 Hz), 9.55 (1H, d, J=0.77 Hz),
9.73 (1H, s), 9.74 (1H, s); MS (ESI) m/z 323/325 (M-H).sup.-,
325/327 (M+H).sup.+.
[0230] Anal. for C.sub.19H.sub.13ClO.sub.3:
[0231] Calc'd: C, 70.27; H, 4.03
[0232] Found: C, 69.80; H, 3.88
EXAMPLE 51
2,3-Dichloro-3'-fluoro-4'-methoxy-1,1'-biphenyl-4-carbaldehyde
[0233] The title compound was prepared by reacting
trifluoro-methanesulfonic acid 2,3-dichloro-4-formyl-phenyl ester
(1.90 g, 5.90 mmol) with 21 (1.30 g, 7.67 mmol) according to method
B to yield 0.84 g (47%) of white solid: mp 160-164.degree. C.;
.sup.1H NMR (DMDO-d.sub.6): .delta. 3.90 (3H, m), 7.28-7.30 (2H,
m), 7.41-7.43 (1H, m), 7.57 (1H, d, J=8.30 Hz), 7.87 (1H, d, J=7.81
Hz), 10.35 (1H, s); MS (EI) m/z 298/300/302 (M).sup.+.
[0234] Anal. for C.sub.14H.sub.9Cl.sub.2FO.sub.2:
[0235] Calc'd: C, 56.21; H, 3.03.
[0236] Found: C, 57.55; H, 2.97.
EXAMPLES 52 AND 53
[0237]
2,3-Dichloro-3'-fluoro-4'-methoxy-1,1'-biphenyl-4-carbaldehyde
(0.72 g, 2.42 mmol) was reacted with boron tribromide (7.25 ml of
1N solution in CH.sub.2Cl.sub.2, 7.25 mmol) according to method E
to produce the following two compounds:
2',3'-Dichloro-4'-(dibromomethyl)-3-fluoro-1,1'-biphenyl-4-ol
(52)
[0238] 130 mg (13%) of grey thick syrup: .sup.1H NMR
(DMSO-d.sub.6): .delta. 7.04 (1H, t, J=8.54 Hz), 7.11 (1H, dd,
J=8.41 Hz, J=1.97 Hz), 7.32 (1H, dd, J=12.18 Hz, J=1.94 Hz), 7.51
(1H, d, J=8.27 Hz), 7.54 (1H, s), 7.95 (1H, d, J=8.23 Hz), 10.23
(1H, s); MS (ESI) m/z 425/427/429 (M-H).sup.-.
[0239] Anal. for C.sub.13H.sub.7Br.sub.2Cl.sub.2FO:
[0240] Calc'd: C, 36.40; H, 1.65.
[0241] Found: C, 37.60; H, 1.69.
2,3-Dichloro-3'-fluoro-4'-hydroxy-1,1'-biphenyl-4-carbaldehyde
(53)
[0242] 140 mg (20%) of white solid: mp 170-171.degree. C.; .sup.1H
NMR (DMSO-d.sub.6): .delta. 7.07 (1H, t, J=8.60 Hz), 7.15 (1H, dd,
J=8.40 Hz, J=1.79 Hz), 7.35 (1H, dd, J=12.15 Hz, J=1.87 Hz), 7.56
(1H, d, J=7.97 Hz), 7.86 (1H, d, J=8.09 Hz), 10.31 (1H, s), 10.35
(1H, s); MS (ESI) m/z 283/285/287 (M-H).sup.-.
[0243] Anal. for C.sub.13H.sub.7Cl.sub.2FO.sub.2:
[0244] Calc'd: C, 54.77; H, 2.47.
[0245] Found: C, 54.93; H, 2.18.
EXAMPLE 54
3,5-Dichloro-4'-hydroxy-1,1'-biphenyl-4-carbaldehyde oxime
[0246] The title compound was prepared by reacting
3,5-dichloro-4'-hydroxy-1,1'-biphenyl-4-carbaldehyde (170 mg, 0.637
mmol) with hydroxylamine hydrochloride (89 mg, 1.27 mmol) according
to Method F to yield 160 mg (89%) of a white solid: mp
183-186.degree. C.; .sup.1H NMR (DMSO-d.sub.6): .delta. 6.86 (2H,
d, J=8.79 Hz), 7.63 (2H, d, J=8.79 Hz), 7.76 (2H, s), 8.25 (1H, s),
9.81 (1H, s), 11.78 (1H, s); MS (ESI) m/z 280/282/284 (M-H).sup.-,
282/284/286 (M+H).sup.+.
EXAMPLE 55
3,5-Dichloro-3'-fluoro-4'-hydroxy-1,1'-biphenyl-4-carbaldehyde
oxime
[0247] The title compound was prepared by reacting
3,5-dichloro-3'-fluoro-4'-hydroxy-1,1'-biphenyl-4-carbaldehyde (290
mg, mmol) with hydroxylamine hydrochloride (140 mg, 1.27 mmol)
according to Method F to yield 260 mg (85%) of a white solid: mp
185-190.degree. C.; .sup.1H NMR (DMSO-d.sub.6): .delta. 7.03 (1H,
t, J=8.86 Hz), 7.46-7.49 (1H, m), 7.68 (1H, dd, J=12.74 Hz, J=2.26
Hz), 7.82 (2H, s), 8.25 (1H, s), 10.24 (1H, s), 11.80 (1H, s); MS
(ESI) m/z 298/300/302 (M-H).sup.-, 300/302/304 (M+H).sup.+.
[0248] Anal. for C.sub.13H.sub.8Cl.sub.2FNO.sub.2. 0.2H.sub.2O:
[0249] Calc'd: C, 51.41; H, 2.79N, 4.61.
[0250] Found: C, 51.70; H, 2.75 N, 4.21.
EXAMPLE 56
2,3-Dichloro-4'-hydroxy-1,1'-biphenyl-4-carbaldehyde oxime
[0251] The title compound was prepared by reacting 48 (140 mg,
0.526 mmol) with hydroxylamine hydrochloride (110 mg, 1.58 mmol)
according to method F to yield 148 mg (100%) of off white solid: mp
208-210.degree. C.; .sup.1H NMR (DMSO-d.sub.6): .delta. 6.84-6.87
(2H, m), 7.26-7.29 (2H, m), 7.36 (1H, d, J=7.94 Hz). 7.80 (1H, d,
J=8.20 Hz), 8.41 (1H, s), 9.72 (1H, s), 11.83 (1H, s); MS (ESI) m/z
280/282/284 (M-H).sup.-, 282/284/286 (M+H).sup.+.
[0252] Anal. for C.sub.13H.sub.9Cl.sub.2NO.sub.2:
[0253] Calc'd: C, 55.35; H, 3.22 N, 4.96.
[0254] Found: C, 55.78; H, 3.59 N, 4.44.
EXAMPLE 57
[0255] 52 (85 mg, 0.20 mmol) was reacted with hydroxylamine
hydrochloride (376 mg, 5.39 mmol) and pyridine (0.43 ml, 5.34 mmol)
for 8 days according to method C to produce the following two
compounds:
2,3-Dichloro-3'-fluoro-4'-hydroxy-1,1'-biphenyl-4-carbaldehyde
oxime (57)
[0256] 17.6 mg (30%) of white solid: mp 225-227.degree. C.; .sup.1H
NMR (DMSO-d.sub.6): .delta. 7.04 (1H, t, J=8.54 Hz), 7.10 (1H, dd,
J=8.35 Hz, J=1.88 Hz), 7.28 (1H, dd, J=12.22 Hz, J=2.01 Hz), 7.39
(1H, d, J=8.14 Hz), 7.81 (1H, d, J=8.15 Hz), 8.41 (1H, s), 10.18
(1H, s), 11.87 (1H, s); MS (ESI) m/z 298/300/302 (M-H).sup.-,
300/302/304 (M+H).sup.+.
[0257] Anal. for C.sub.13H.sub.8Cl.sub.2FNO.sub.2. 0.09 TFA:
[0258] Calc'd: C, 51.00; H, 2.63 N, 4.51.
[0259] Found: C, 50.97; H, 2.37 N, 4.33.
Methyl
2,3-dichloro-3'-fluoro-4'-hydroxy-1,1'-biphenyl-4-carboxylate
[0260] 16.9 mg (27%) of white solid: mp 152-154.degree. C.; .sup.1H
NMR (DMSO-d.sub.6): .delta. 3.90 (3H, s), 7.05 (1H, t, J=8.54 Hz),
7.11 (1H, dd, J=8.41H, J=1.55 Hz), 7.31 (1H, dd, J=12.16 Hz, J=1.68
Hz), 7.47 (1H, d, J=8.02 Hz), 7.76 (1H, d, J=8.64 Hz); MS (ESI) m/z
313/315/317 (M+H).sup.+.
[0261] Anal. for C.sub.14H.sub.9Cl.sub.2FO.sub.3:
[0262] Calc'd: C, 53.36; H, 2.88.
[0263] Found: C, 51.94; H, 2.54.
EXAMPLE 58
Table 1. 4'-Hydroxy-Biphenyl-carbaldehyde Oxime Derivatives
[0264] TABLE-US-00001 ##STR22## Ex. R.sup.1 R.sup.2 R.sup.3 R.sup.5
R.sup.6 R.sup.7 ER.sub..beta. (nM) ER.sub..alpha. (nM) 29 H Me H H
H H 163 3193 30 H H H H H H 3840 >5000 31 H Cl H H H H 85 1540
32 H H F H H H 421 3450 33 H F H H H H 161 1720 34 H H Cl H H H 98
398 35 H OMe H H H H 1058 7990 36 H H Me H H H 585 2821 37 H Cl H H
F H 54 1910 38 H Cl H H F F 268 >7000 39 H Cl H Me H H 27 174 54
Cl Cl H H H H 8 64 55 Cl Cl H H F H 9 270 56 H Cl Cl H H H 49 851
57 H Cl Cl H F H 81 1957
[0265] The results obtained in the standard pharmacologic test
procedure demonstrate that the compounds of this invention are
estrogenic compounds, some with strong preferential affinity for
the ER.beta. receptor. The compounds of this invention range from
having high preferential affinity for ER.beta. over ER.alpha. to
almost equal affinity for both receptors. Thus, compounds of this
invention will span a range of activity based, at least partially,
on their receptor affinity selectivity profiles. Additionally,
because each novel receptor ligand complex is unique and thus its
interaction with various coregulatory proteins is unique, compounds
of this invention will display different modulatory behavior
depending on the cellular context they are in. For example, in some
cell-types, it is possible for a compound to behave as an estrogen
agonist while in other tissues, an antagonist. Compounds with such
activity have sometimes been referred to as SERMs (Selective
Estrogen Receptor Modulators). Unlike many estrogens, however, many
of the SERMs do not cause increases in uterine wet weight. These
compounds are antiestrogenic in the uterus and can completely
antagonize the trophic effects of estrogen agonists in uterine
tissue. These compounds, however, act as estrogen agonists in the
bone, cardiovascular, and central nervous systems. Due to this
tissue selective nature of these compounds, they are useful in
treating or preventing in a mammal disease states or syndromes
which are caused or associated with an estrogen deficiency (in
certain tissues such as bone or cardiovascular) or an excess of
estrogen (in the uterus or mammary glands).
[0266] Even beyond such cell-specific modulation, compounds of this
invention also have the potential to behave as agonists on one
receptor type while behaving as antagonists on the other. For
example, it has been demonstrated that compounds can be an
antagonist on ER.beta. while being an agonist on ER.alpha. (Meyers,
Marvin J.; Sun, Jun; Carlson, Kathryn E.; Katzenellenbogen, Benita
S.; Katzenellenbogen, John A. J. Med. Chem. (1999), 42(13),
2456-2468). Such ERSAA (Estrogen Receptor Selective Agonist
Antagonist) activity provides for pharmacologically distinct
estrogenic activity within this series of compounds.
[0267] Standard pharmacological test procedures are readily
available to determine the activity profile of a given test
compound. The following examples briefly summarize several
representative test procedures. Standard pharmacological test
procedures for SERMs are also provided in U.S. Pat. Nos. 4,418,068
and 5,998,402.
EXAMPLE 59
Rat Uterotrophic/Antiuterotrophic Test Procedure
[0268] The estrogenic and antiestrogenic properties of the
compounds can be determined in an immature rat uterotrophic assay
(4 day) that (as described previously by L. J. Black and R. L.
Goode, Life Sciences, 26, 1453 (1980). Immature Sprague-Dawley rats
(female, 18 days old) were tested in groups of six. The animals are
treated by daily ip injection with 10 uG compound, 100 uG compound,
(100 uG compound+1 uG 17.beta.-estradiol) to check
antiestrogenicity, and 1 uG 17.beta.-estradiol, with 50% DMSO/50%
saline as the injection vehicle. On day 4 the animals are
sacrificed by CO.sub.2 asphyxiation and their uteri removed and
stripped of excess lipid, any fluid removed and the wet weight
determined. A small section of one horn is submitted for histology
and the remainder used to isolate total RNA in order to evaluate
complement component 3 gene expression.
EXAMPLE 60
6-Week Ovariectomized Rat Test Procedure--Bone and
Cardioprotection
[0269] Female Sprague Dawley CD rats, ovx or sham ovx, are obtained
1 day after surgery from Taconic Farm (weight range 240-275 g).
They are housed 3 or 4 rats/cage in a room on a 12/12 (light/dark)
schedule and provided with food (Purina 5K96C rat chow) and water
ad libitum. Treatment for all studies begin 1 day after the animals
arrival and dosed 7 days per week as indicated for 6 weeks. A group
of age matched sham operated rats not receiving any treatment serve
as an intact, estrogen replete control group for each study.
[0270] All treatments are prepared in 1% tween 80 in normal saline
at defined concentrations so that the treatment volume is 0.1
mL/100 g body weight. 17.beta.-estradiol is dissolved in corn oil
(20 .mu.g/mL) and delivered subcutaneously, 0.1 mL/rat. All dosages
are adjusted at three week intervals according to group mean body
weight measurements.
[0271] Five weeks after the initiation of treatment and one week
prior to the termination of the study, each rat is evaluated for
bone mineral density (BMD). The total and trabecular density of the
proximal tibia are evaluated in anesthetized rats using an XCT-960M
(pQCT; Stratec Medizintechnik, Pforzheim, Germany). The
measurements are performed as follows: Fifteen minutes prior to
scanning, each rat is anesthetized with an intraperitoneal
injection of 45 mg/kg ketamine, 8.5 mg/kg xylazine, and 1.5 mg/kg
acepromazine.
[0272] The right hind limb is passed through a polycarbonate tube
with a diameter of 25 mm and taped to an acrylic frame with the
ankle joint at a 90.degree. angle and the knee joint at
180.degree.. The polycarbonate tube is affixed to a sliding
platform that maintains it perpendicular to the aperture of the
pQCT. The platform is adjusted so that the distal end of the femur
and the proximal end of the tibia would be in the scanning field. A
two dimensional scout view is run for a length of 10 mm and a line
resolution of 0.2 mm. After the scout view is displayed on the
monitor, the proximal end of the tibia is located. The pQCT scan is
initiated 3.4 mm distal from this point. The pQCT scan is 1 mm
thick, has a voxel (three dimensional pixel) size of 0.140 mm, and
consists of 145 projections through the slice.
[0273] After the pQCT scan is completed, the image is displayed on
the monitor. A region of interest including the tibia but excluding
the fibula is outlined. The soft tissue is automatically removed
using an iterative algorithm. The density of the remaining bone
(total density) is reported in mg/cm.sup.3. The outer 55% of the
bone is peeled away in a concentric spiral. The density of the
remaining bone (Trabecular density) is reported in mg/cm.sup.3. One
week after BMD evaluation the rats are euthanized by carbon dioxide
suffocation and blood collected for cholesterol determination. The
uteri are removed and the weights taken. Total cholesterol is
determined using a Boehringer-Mannheim Hitachi 911 clinical
analyzer using the Cholesterol/HP kit. Statistics were compared
using one-way analysis of variance with Dunnet's test.
EXAMPLE 61
MCF-7/ERE Antiproliferative Test Procedure
[0274] Stock solutions of test compounds (usually 0.1 M) are
prepared in DMSO and then diluted 10 to 100-fold with DMSO to make
working solutions of 1 or 10 mM. The DMSO stocks are stored at
either 4.degree. C. (0.1 M) or -20.degree. C. (<0.1M). MCF-7
cells are passaged twice a week with growth medium [D-MEM/F-12
medium containing 10% (v/v) heat-inactivated fetal bovine serum, 1%
(v/v) Penicillin-Streptomycin, and 2 mM glutaMax-1]. The cells are
maintained in vented flasks at 37.degree. C. inside a 5%
CO.sub.2/95% humidified air incubator. One day prior to treatment,
the cells are plated with growth medium at 25,000/well into 96 well
plates and incubated at 37.degree. C. overnight.
[0275] The cells are infected for 2 hr at 37.degree. C. with 50
.mu.l/well of a 1:10 dilution of adenovirus 5-ERE-tk-luciferase in
experimental medium [phenol red-free D-MEM/F-12 medium containing
10% (v/v) heat-inactived charcoal-stripped fetal bovine serum, 1%
(v/v) Penicillin-Streptomycin, 2 mM glutaMax-1, 1 mM sodium
pyruvate]. The wells are then washed once with 150 .mu..lamda. of
experimental medium. Finally, the cells are treated for 24 hr at
37.degree. C. in replicates of 8 wells/treatment with 150
.mu..lamda./well of vehicle (.ltoreq.0.1% v/v DMSO) or compound
that is diluted .gtoreq.1000-fold into experimental medium.
[0276] Initial screening of test compounds is done at a single dose
of 1 .mu.M that is tested alone (agonist mode) or in combination
with 0.1 nM 17.beta.-estradiol (EC.sub.80; antagonist mode). Each
96 well plate also includes a vehicle control group (0.1% v/v DMSO)
and an agonist control group (either 0.1 or 1 nM
17.beta.-estradiol). Dose-response experiments are performed in
either the agonist and/or antagonist modes on active compounds in
log increases from 10.sup.-14 to 10.sup.-5 M. From these
dose-response curves, EC.sub.50 and IC.sub.50 values, respectively,
are generated. The final well in each treatment group contains 5
.mu.l of 3.times.10.sup.-5 M ICI-182,780 (10.sup.-6 M final
concentration) as an ER antagonist control.
[0277] After treatment, the cells are lysed on a shaker for 15 min
with 25 .mu.l/well of 1.times. cell culture lysis reagent (Promega
Corporation). The cell lysates (20 .mu.l) are transferred to a 96
well luminometer plate, and luciferase activity is measured in a
MicroLumat LB 96 P luminometer (EG & G Berthold) using 100
.mu.l/well of luciferase substrate (Promega Corporation). Prior to
the injection of substrate, a 1 second background measurement is
made for each well. Following the injection of substrate,
luciferase activity is measured for 10 seconds after a 1 second
delay. The data are transferred from the luminometer to a Macintosh
personal computer and analyzed using the JMP software (SAS
Institute); this program subtracts the background reading from the
luciferase measurement for each well and then determines the mean
and standard deviation of each treatment.
[0278] The luciferase data are transformed by logarithms, and the
Huber M-estimator is used to down-weight the outlying transformed
observations. The JMP software is used to analyze the transformed
and weighted data for one-way ANOVA (Dunnett's test). The compound
treatments are compared to the vehicle control results in the
agonist mode, or the positive agonist control results (0.1 nM
17.beta.-estradiol) in the antagonist mode. For the initial single
dose experiment, if the compound treatment results are
significantly different from the appropriate control (p<0.05),
then the results are reported as the percent relative to the
17.beta.-estradiol control [i.e., ((compound-vehicle
control)/(17.beta.-estradiol control-vehicle control)).times.[00].
The JMP software is also used to determine the EC.sub.50 and/or
IC.sub.50 values from the non-linear dose-response curves.
EXAMPLE 62
Inhibition of LDL Oxidation-Antioxidant Activity
[0279] Porcine aortas are obtained from an abattoir, washed,
transported in chilled PBS, and aortic endothelial cells are
harvested. To harvest the cells, the intercostal vessels of the
aorta are tied off and one end of the aorta clamped. Fresh, sterile
filtered, 0.2% collagenase (Sigma Type I) is placed in the vessel
and the other end of the vessel then clamped to form a closed
system. The aorta is incubated at 37.degree. C. for 15-20 minutes,
after which the collagenase solution is collected and centrifuged
for 5 minutes at 2000.times.g. Each pellet is suspended in 7 mL of
endothelial cell culture medium consisting of phenol red free
DMEM/Ham's F12 media supplemented with charcoal stripped FBS (5%),
NuSerum (5%), L-glutamine (4 mM), penicillin-streptomycin (1000
U/ml, 100 .mu.g/ml) and gentimicin (75 .mu.g/ml), seeded in 100 mm
petri dish and incubated at 37.degree. C. in 5% CO.sub.2. After 20
minutes, the cells are rinsed with PBS and fresh medium added, this
was repeated again at 24 hours. The cells are confluent after
approximately 1 week. The endothelial cells are routinely fed twice
a week and, when confluent, trypsinized and seeded at a 1:7 ratio.
Cell mediated oxidation of 12.5 .mu.g/mL LDL is allowed to proceed
in the presence of the compound to be evaluated (5 .mu.M) for 4
hours at 37.degree. C. Results are expressed as the percent
inhibition of the oxidative process as measured by the TBARS
(thiobarbituric acid reactive substances) method for analysis of
free aldehydes (Yagi K., Biochem Med 15:212-216 (1976)).
EXAMPLE 63
D12 Hypothalmic Cell Test Procedure
[0280] D12 rat hypothalamic cells are subcloned from the RCF.sub.17
parental cell line and stored frozen. They are routinely grown in
DMEM:F12 (1:1), glutaMAX-1 (2 mM), penicillin (100
U/ml)-streptomycin (100 mg/ml), plus 10% fetal bovine serum (FBS).
The cells are plated in phenol red-free medium (DMEM:F12, glutaMAX,
penicillin-streptomycin) containing 2-10% charcoal stripped FBS at
a subconfluent density (1-4.times.10 6 cells/150 mm dish). The
cells are refed 24 h later with medium containing 2% stripped
serum. To test for agonist activity, cells are treated with 10 nM
17.beta.-estradiol or various doses of test compound (1 mM or a
range from 1 pM to 1 mM). To test for antagonist activity the cells
are treated with 0.1 nM 17.beta.-estradiol in the absence or
presence of varying doses (100 pM to 1 mM) of test compound.
Control dishes are also treated with DMSO as a negative control.
Forty-eight hours after hormone addition, the cells are lysed and
binding test procedure performed.
[0281] For each binding test procedure 100-150 mg protein is
incubated with 10 nM .sup.3H-R5020+100-fold excess R5020 in a 150
ml volume. Triplicate reactions (three with R5020, three without
R5020) are prepared in a 96 well plate. The protein extract is
added first followed by .sup.3H-R5020 or .sup.3H-R5020+100.times.
unlabeled R5020. The reaction is performed for 1-2 hr at room
temperature. The reaction is stopped by the addition of 100 ml cold
5% charcoal (Norit SX-4), 0.5% dextran 69K (Pharmacia) in TE pH
7.4. After 5 min at room temperature, the bound and unbound ligand
are separated by centrifugation (5 min, 1000 RCF, 4.degree. C.).
The supernatant solution (.about.150 ml) is removed and transferred
to a scintillation vial. Following the addition of scintillation
fluid (Beckman Ready Protein+), the samples are counted for 1 min
in a scintillation counter.
EXAMPLE 64
Progesterone Receptor in the CNS Preoptic Area
[0282] Sixty (60) day old female Sprague-Dawley rats are
ovariectomized. The animals are housed in an animal care facility
with a 12-h light, 12-h dark photoperiod and free access to tap
water and rodent chow.
[0283] Ovariectomized animals are randomly divided into groups that
are injected with vehicle (50% DMSO, 40% PBS, 10% ethanol vehicle),
17.beta.-estradiol (200 ng/kg) or the compound to be tested.
Additional animals are injected with the test compound 1 hr prior
to injection of 17.beta.-estradiol to evaluate the antagonistic
properties of this compound. Six hrs after s.c. injection, animals
are euthanized with a lethal dose of CO.sub.2 and their brains
collected and frozen.
[0284] Tissue collected from animals is cut on a cryostat at
-16.degree. C. and collected on Silane-coated microscope slides.
The section-mounted slides are then dried on a slide warmer
maintained at 42.degree. C. and stored in desiccated slide boxes at
-80.degree. C. Prior to processing, the desiccated slide boxes are
slowly warmed to room temperature (-20.degree. C. for 12-16 hrs;
4.degree. C. for 2 hrs; room temperature for 1 hr) to eliminate
condensation formation on slides and thus minimize tissue and RNA
degradation. The dry slides are loaded into metal racks, postfixed
in 4% paraformaldehyde (pH 9.0) for 5 min and processed as
previously described.
[0285] A plasmid containing a 815 bp fragment of the rat PR cDNA 9
(ligand binding domain) is linearized and used to generate a S
35-UTP labeled probe that is complimentary to a portion of the rat
PR mRNA. Processed section-mounted slides are hybridized with 20
.mu.ml of hybridization mix containing the riboprobe (4-6.times.10
6 DPM/slide) and 50% formamide and incubated overnight in a
55.degree. C. humidified chamber. In the morning, the slides are
placed in metal racks that are immersed in 2.times.SSC (0.15 M
NaCl, 0.015 M sodium citrate; pH 7.0)/10 mM DTT. The racks are all
transferred to a large container and washed in 2.times.SSC/10 mM
DTT for 15 min at RT with gentle agitation. Slides are then washed
in RNase buffer at 37.degree. C. for 30 min, treated with RNase A
(2 mg/ml) for 30 min at 37.degree. C., and washed for 15 min in
room temperature 1.times.SSC. Subsequently, the slides are washed
(2.times.30 min) in 65.degree. C. in 0.1.times.SSC to remove
nonspecific label, rinsed in room temperature 0.1.times.SSC for 15
min and dehydrated with a graded series of alcohol: ammonium
acetate (70%, 95%, and 100%). Air dried slides are opposed to x-ray
film for 3 days and then photographically processed. The slides
from all animals are hybridized, washed, exposed and
photographically processed together to eliminate differences due to
interassay variation in conditions.
EXAMPLE 65
Rat Hot Flush--CNS Effects
[0286] Ovariectomized-female, 60 day-old Sprague-Dawley rats are
obtained following surgery. The surgeries are done a minimum of 8
days prior to the first treatment. The animals are housed
individually under 12 h light/dark cycle and given standard rat
chow and water ad libitum.
[0287] Two control groups are included in every study. Doses are
prepared based on mg/kg mean group body weight in either 10% DMSO
in sesame oil (sc studies) or in 1.0% tween 80 in saline (po
studies). Animals are administered test compounds at doses ranging
from 0.01 to 10 mg/kg mean group body weight. Vehicle and ethinyl
estradiol (EE) controls (0.1 mg/kg, sc or 0.3 mg/kg, po) control
groups are included in each test. When the compounds are tested for
their antagonist activity, EE is coadministered at 0.1 or 0.3 mg/kg
for sc or po studies, respectively. The test compounds are
administered up to the day tail skin temperature is measured.
[0288] After the acclimation period of four days, the animals are
treated once daily with the compound(s) of interest. There are 10
animals/treatment group. Administration of the compound is either
by sc injection of 0.1 ml in the nape of the neck or po in a volume
of 0.5 ml. On the 3rd day of treatment, a morphine pellet (75 mg
morphine sulfate) is implanted subcutaneously. On the 5th day of
treatment, one or two additional morphine pellets are implanted. On
the eighth day, approximately half of the animals are injected with
Ketamine (80 mg/kg, intramuscularly) and a thermocouple, connected
with to a MacLab Data Acquisition System (API Instruments, Milford,
Mass.) is taped on the tail approximately one inch from the root of
the tail. This system allowed the continuous measurement of tail
skin temperature. Baseline temperature is measured for 15 min, then
naloxone (1.0 mg/kg) is given sc (0.2 ml) to block the effect of
morphine and tail skin temperature is measured for one hour
thereafter. On the ninth day, the remaining of the animals are set
up and analyzed similarly.
EXAMPLE 66
Vasomotor Function in Isolated Rat Aortic Rings
[0289] Sprage-Dawley rats (240-260 grams) are divided into 4
groups:
1. Normal non-ovariectomized (intact)
2. Ovariectomized (ovex) vehicle treated
3. Ovariectomized 17.beta.-estradiol treated (1 mg/kg/day)
4. Ovariectomized animals treated with test compound (I.e., 1
mg/kg/day)
[0290] Animals are ovariectomized approximately 3 weeks prior to
treatment. Each animal receives 1 mg/kg/day of either
17.beta.-estradiol sulfate or test compound suspended in distilled,
deionized water with 1% tween-80 by gastric gavage. Vehicle treated
animals received an appropriate volume of the vehicle used in the
drug treated groups.
[0291] Animals are euthanized by CO.sub.2 inhalation and
exsanguination. Their thoracic aortas are rapidly removed and
placed in 37.degree. C. physiological solution with the following
composition (mM): NaCl (54.7), KCl (5.0), NaHCO.sub.3 (25.0),
MgCl.sub.2 2H.sub.2O (2.5), D-glucose (11.8) and CaCl.sub.2 (0.2)
gassed with CO.sub.2--O.sub.2, 95%/5% for a final pH of 7.4. The
advantitia is removed from the outer surface and the vessel is cut
into 2-3 mm wide rings. Rings are suspended in at 10 mL tissue bath
with one end attached to the bottom of the bath and the other to a
force transducer. A resting tension of 1 gram is placed on the
rings. Rings are equilibrated for 1 hour, signals are acquired and
analyzed.
[0292] After equilibration, the rings are exposed to increasing
concentrations of phenylephrine (10.sup.-8 to 10.sup.-4 M) and the
tension recorded. Baths are then rinsed 3 times with fresh buffer.
After washout, 200 mM L-NAME is added to the tissue bath and
equilibrated for 30 minutes. The phenylephrine concentration
response curve is then repeated.
EXAMPLE 67
Eight Arm Radial Arm Maze--Cognition Enhancement
[0293] Male Sprague-Dawley, CD rats (Charles River, Kingston, N.Y.)
weighing 200-250 g on arrival are used. For one week, the rats are
housed, six per cage, with standard laboratory chow and water
available ad libitum. Housing is in a colony room maintained at
22.degree. C. and had a 12 hour light/dark cycle with lights on at
6:00 AM. Following habituation to the facility, animals are
individually housed and maintained at 85% of free-feeding weight.
Once stable weights are attained, the rats are acclimated to the
8-arm radial maze.
[0294] The structure of the maze is an adaptation from that of
Peele and Baron (Pharmacology, Biochemistry, and Behavior,
29:143-150, 1988). The maze is elevated to a height of 75.5 cm and
composed of a circular area surrounded by 8 arms radiating away
from the center, equidistant from one another. Each arm is 58 cm
long.times.13 cm high. A clear plexiglass cylinder is oared to
enclose the animal in the center portion of the maze prior to the
start of each session. Each arm of the maze is equipped with 3 sets
of photocells interfaced to a data acquisition unit, which in turn
is interfaced to a computer. The photocells are used to track the
movement of the rat in the maze. Pellet feeders located above food
cups at the end of each arm, dispensed two 45 mg chocolate pellets
when the outer photocell of the arm is activated for the first time
in a given session. The maze is located in a testing room with
black and white geometric posters on each wall to serve as visual
cues. During all training and testing procedures, white noise is
audible (.about.70 db).
[0295] The training procedure consists of five phases, each with
daily sessions lasting 5 or 10 minutes. A 10 second delay is
imposed between the time the rat is placed in the center portion of
the maze and when the cylinder is raised to begin the session.
During Phase 1, food-restricted pairs of rats are placed on the
maze for 10 minutes with 45 mg chocolate food pellets scattered
throughout the 8 arms of the maze. During Phase II, each rat is
placed individually on the maze for a 10 minute period, with
pellets scattered from the middle photocell to the food cup of each
arm. During Phase III, each rat is placed on the maze for a 10
minute period, with food pellets located only in and around the
food cups in each arm. In Phase IV, each rat is allowed 10 minutes
to collect two pellets from each arm. Re-entry into an arm is
considered an error. Rats are trained daily in this manner until
they achieved criterion performance with less than or equal to 2
total errors on three consecutive days of training. Total
habituation and training time is approximately 3 weeks.
[0296] Test compound is prepared in phosphate buffered saline and
administered in a volume of 1 ml/kg. Scopolamine HBr (0.3 mg/kg
s.c.) served as the impairing agent, producing an increase in error
rate (loss of memory). Test compound is given intraperitoneally
simultaneously with scopolamine, 30 minutes prior to the first maze
exposure on any given test day.
[0297] To assess the test compound, an 8.times.8 balanced latin
square for repeated measures is designed, in order to achieve a
high experimental efficiency with the least amount of animals.
Eight experimental sessions, two per week, are conducted with the 8
treatments (vehicle, scopolamine, 3 doses of test compound in
combination with scopolamine) randomized within each session. Each
treatment followed every other treatment the same number of times.
Therefore, the residual effect of every treatment could be
estimated and removed from the direct treatment effect. Following
ANOVA, multiple comparisons are performed using Dunnett's two-sided
test on adjusted means.
[0298] Animals that did not make 4 correct choices within 5 minutes
during the first exposure, or that had not made a total of 8
choices by the end of the 2nd exposure, are considered to have
"timed-out" for that session. Any animal that "timed-out" following
administration of more than one dose of the test compound is
excluded from the analysis.
EXAMPLE 68
Neuroprotection
[0299] Inhibition of Time-Dependent Death of Cells in Primary
Cortical Neuron Cultures
[0300] Primary cortical neurons were produced from rat brains that
were 0-1 day old using a variation of methods described by Monyer
et al. 1989, Brain Research 483:347-354. Dispersed brain tissue was
grown in DMEM/10% PDHS (pregnant donor horse serum) for three days
and then treated with cytosine arabinoside (ARC) for two days to
remove contaminating glial cells. On day 5, the ARC media was
removed and replaced with DMEM/10% PDHS. The neuronal cells were
cultured for a further 4-7 days before use.
[0301] Control primary neuronal cultures show progressive cell
death between days 12 and 18 in culture. Twelve cultures were
evaluated on days 12 and 16 for levels of the enzyme lactate
dehydrogenase (LD) after adding test compound to 6 cultures
maintained in DMEM and 10% PDHS on day 9 and maintaining the
remaining cultures as controls. LD was assayed using a variation of
the method by Wroblewski et al. 1955, Proc. Soc. Exp. Biol. Med.
90:210-213. LD is a cytosolic enzyme which is commonly used in both
clinical and basic research to determine tissue viability. An
increase in media LD is directly related to cell death.
[0302] Neuroprotection Against Cytotoxicity Induced by
Hypoglycemia
[0303] C6 glioma cells obtained from ATCC were plated in RPMI media
with FBS at a concentration of 1.times.10<6> cells/ml in
FALCON 25 cm.sup.2 tissue culture flasks. Four hours prior to the
onset of hypoglycemia, the maintenance media was discarded,
monolayers were washed twice in the appropriate media and then
incubated for four hours at 37.degree. C. in either serum free or
serum free plus test compound. Kreb's Ringer Phosphate buffer was
used to wash the monolayers twice before the addition of
appropriate glucose treatment. RPMI medium contains 2 mg
glucose/ml; flasks were divided into groups of 6 each receiving
100% glucose (2 mg/ml), 80% glucose (1.6 mg/ml), 60% glucose (1.2
mg/ml) or 0% glucose (buffer) or supplemented with test compound.
All flasks were incubated for 20 hours and then evaluated for
total, live, and dead cell number utilizing trypan blue.
[0304] Neuroprotection Against Excitotoxic Amino Acids
[0305] Five culture dishes containing SK-N-SH neuroblastoma cells
were treated with test compound and 5 culture dishes were treated
with RPMI media. Four hours later, all cell were treated with NMDA
(500 mu M) for 5 minutes. Total live cells and dead cells were then
determined.
[0306] Neuroprotection Against Oxygen-Glucose Deprivation
[0307] Analysis of pyknotic nuclei to measure apoptosis: Cortical
neurons are prepared from E18 rat fetus and plated in 8-well
chamber slides precoated with poly-D-lysine (10 ng/ml) and serum at
a density of 100,000 cells/well. Cells are plated in high glucose
DMEM containing 10% FCS and kept in the incubator at 37.degree. C.
with 10% CO.sub.2/90% air. On the next day, serum is removed by
replacing culture media with high glucose DMEM containing B27
supplement and cells are kept in the incubator without further
media change until the day of experiment. On day 6, slides are
divided into two groups; control group and OGD group. Cells in
control group receive DMEM with glucose and custom B27 (without
antioxidants). Cells in OGD group receive no-glucose DMEM with
custom B27, which has been degassed under vacuum for 15 min. Cells
are flushed with 90% N.sub.2/10% CO.sub.2 for 10 min in an airtight
chamber and incubated at 37.degree. C. for 6 hrs. After 6 hrs, both
control and OGD cells are subject to replacement of media
containing either vehicle (DMSO) or test compound in
glucose-containing DMEM with custom B27. Cells are returned to
normoxic incubator at 37.degree. C. After 24 hrs, cells are fixed
in 4% PFA for 10 min at 4.degree. C. and stained with Topro
(Fluorescent nuclear binding dye). Apoptosis is assessed using
Laser Scanning Cytometer by measuring pyknotic nuclei.
[0308] Measurement of LDH release as an indication of cell death:
Cortical neurons are prepared from E18 rat fetus and plated in
48-well culture plates precoated with poly-D-lysine (10 ng/ml) and
serum at a density of 150,000 cells/well. Cells are plated in high
glucose DMEM containing 10% FCS and kept in the incubator at
37.degree. C. with 10% CO.sub.2/90% air. On the next day, serum is
removed by replacing culture media with high glucose DMEM
containing B27 supplement. On day 6, cells are divided into two
groups; control group and OGD group. Cells in control group receive
DMEM with glucose and custom B27 (without antioxidants). Cells in
OGD group receive no-glucose DMEM with custom B27, which has been
degassed under vacuum for 15 min. Cells are flushed with 90%
N.sub.2/10% CO.sub.2 for 10 min in an airtight chamber and
incubated at 37.degree. C. for 6 hrs. After 6 hrs, both control and
OGD cells are subject to replacement of media containing either
vehicle (DMSO) or test compound in glucose-containing DMEM with
custom B27. Cells are returned to normoxic incubator at 37.degree.
C. After 24 hrs, cell death is assessed by measuring cellular
release of LDH (lactate dehydrogenase) into the culture medium. For
LDH assay, an aliquot of 50 .mu.l culture medium is transferred
into the 96 well plate. After the addition of 140 .mu.l 0.1 M
potassium phosphate buffer (pH 7.5) and 100 .mu.l 0.2 mg/ml NADH,
the plate is let sit in the dark at room temperature for 20 min.
The reaction is initiated by the addition of 10 .mu.l of sodium
pyruvate. The plate is read immediately at 340 nM in a Thermomax
plate reader (Molecular Devices). The absorbance, an index of NADH
concentration, is recorded every 6 seconds for 5 minutes and the
slope indicating the rate of NADH disappearance is used to
calculate LDH activity. LDH
Activity(U/ml)=(.alpha.A/min)(TCF)(20)(0.0833)/(0.78) [0309] where:
0.0833=proportionality constant [0310] 0.78=instrument light path
length (cm)
EXAMPLE 69
HLA Rat Test Procedure--Crohn's Disease and Inflammatory Bowel
Disorders
[0311] Male HLA-B27 rats are obtained from Taconic and provided
unrestricted access to a food (PMI Lab diet 5001) and water. At the
start of the study, rats are 22-26 weeks old.
[0312] Rats are dosed subcutaneously once per day for seven days
with one of the formulations listed below. There are five rats in
each group and the last dose is administered two hours before
euthanasia.
[0313] vehicle (50% DMSO/50% Dulbecco's PBS)
[0314] 17.alpha.-ethinyl-17.beta.-estradiol (10 .mu.g/kg)
[0315] test compound
[0316] Stool quality is observed daily and graded according to the
following scale: Diarrhea=3; soft stool=2; normal stool=1. At the
end of the test procedure, serum is collected and stored at
-70.degree. C. A section of colon is prepared for histological
analysis and an additional segment is analyzed for myeloperoxidase
activity.
[0317] The following method is used to measure myeloperoxidase
activity. Colon tissue is harvested and flash frozen in liquid
nitrogen. A representative sample of the entire colon is used to
ensure consistency between samples. The tissue is stored at
-80.degree. C. until use. Next, the tissue is weighed
(approximately 500 mg) and homogenized in 1:15 w/v of 5 mM H.sub.2
KPO.sub.4 (pH 6) washing buffer. The tissue is spun down at
20,000.times.g in a Sorvall RC 5B centrifuge for 45 minutes at
2-8.degree. C. Supernatant is then discarded. Tissue is resuspended
and homogenized in 2.5 ml (1:5 w/v) of 50 mM H.sub.2 KPO.sub.4 with
10 mM EDTA and 0.5% Hex Ammonium Bromide to help solubilize the
intracellular MPO. Tissue is frozen in liquid Nitrogen, thawed in a
37.degree. C.-water bath and sonicated for 15 seconds to ensure
membrane lysis. This procedure is repeated 3 times. Samples are
then kept on ice for 20 minutes and centrifuged at 12,000.times.g
for 15 minutes at 2-8.degree. C. The supernatant is analyzed
following these steps.
[0318] The test mixture is prepared by adding 2.9 ml of 50 mM
H.sub.2 KPO.sub.4 with 0.167 O-Dianisidine/ml with 0.0005%
H.sub.2O.sub.2 into a reaction tube. When hydrogen peroxide is
degraded, O-Dianisidine is oxidized and absorbs at 460 nm in a
concentration dependent manner. The mixture is heated to 25.degree.
C. One hundred (100) .mu.L of the tissue supernatant is added to
the reaction tube, incubated for one minute at 25.degree. C., then
1 ml is transferred to a disposable plastic cuvette. OD is measured
every 2 minutes reaction time at 460 nm against a blank containing
2.9 ml of the reaction mixture and 100 .mu.l of the 0.5% ammonium
bromide solution.
[0319] Enzyme activity units are quantified by comparison of
absorbence @ 460 to a standard curve prepared with purified human
MPO 31.1 Units/Vial. The MPO is reconstituted and serially diluted
using 50 mM H.sub.2 KPO.sub.4 with 10 mM EDTA and 0.5% Hex Ammonium
Bromide to four known concentrations. Sample absorbencies are
compared against this curve to determine activity.
[0320] Histological analysis is performed as follows. Colonic
tissue is immersed in 10% neutral buffered formalin. Each specimen
of colon is separated into four samples for evaluation. The
formalin-fixed tissues are processed in a vacuum infiltration
processor for paraffin embedding. The samples are sectioned at 5
.mu.m and then stained with hematoxylin and eosin (H&E) for
blinded histologic evaluations using a scale modified after
Boughton-Smith. After the scores are completed the samples are
unblinded, and data are tabulated and analyzed by ANOVA linear
modeling with multiple mean comparisons.
[0321] All patents, publications, and other documents cited herein
are hereby incorporated by reference in their entirety.
* * * * *