This invention relates to Phenanthridine Carbonyl Phenols as Cytokine Modulato
BACKGROUND
The present invention relates generally to phenanthridine carbonyl compounds
such as substituted phenanthridine carbonyl phenols and methods of using them.
[0001] The ability of ligands for the estrogen receptor to inhibit inflammatory
gene expression and cause a reduction of cytokines, chemokines, adhesion molecules and
inflammatory enzymes is well known. A common component of chronic inflammatory
conditions is polymorphonuclear leukocyte and monocyte infiltration into the site of
damage through increased expression of cytokines and adhesion molecules responsible
for their recruitment. Overproduction of the cytokine interleukin (IL-6) has been
associated with states of chronic inflammation (Bauer M. A., Herrmann F., Ann.
Hematol, 1991, 62, 203). Synthesis of the IL-6 gene is believed to be induced by the
transcription factor nuclear factor KB (NF-KB). Interference at this step in the
inflammatory process can effectively regulate the uncontrolled proliferative process that
occu in these chronic conditions.
Activation of the estrogen receptor provides a means to treat the inflammatory
component of diseases such as atherosclerosis, myocardial infarction (MI), congestive
heart failure (CHF), inflammatory bowel disease and arthritis, in part by interfering with
cytokine expression. Other therapeutic indications for these type of molecules include
type II diabetes (Cefalu, J Womens Health & Gender-based Med. 2001, 10, 241; Yuan et
al, Science, 2001, 293, 1673), osteoarthritis (Pelletier et al, Arthr. & Rheum., 2001,
44:1237; Felson et al, Curr Opinion Rheum, 1998, 10, 269) asthma (Chin-Chi Lin et.al.,
Immunol Lett., 2000, 73, 57), Alzheiemer's disease (Roth, A. et. al.,; J. Neurosci. Res.,
1999, 57, 399) and autoimmune diseases such as multiple sclerosis and rheumatoid
arthritis.
In view of the foregoing, there exists a need for the identification of ligands for
the estrogen receptor and for methods of using the identified ligands to modulate the
activity of the receptor and, preferably, treat disease.
SUMMARY
The present invention provides, inter alia, phenanthridine carbonyl compounds
and compositions, particularly those that find use as ligands for the estrogen receptor, and
methods of using them. Representative compounds of the invention include those of
wherein
RI, R2, , and Rt, are, independently, hydrogen, lower alkyl, halogen, or aryl;
R Rg, Rg, and RIO, are, independently, hydrogen, lower alkyl or halogen;
RH, Ri2, RH, and RIS are, independently, hydrogen, hydroxy, lower alkyl, alkoxy,
or halogen;
one of and R is hydrogen and the other is lower alkyl;
RB is hydrogen, -(C=O)Ri6, -S(O)2Ri7, -S(O)2N(Ri8)(Ri9), or D-glucuronidate;
Ri6 is alkyl, aralkyl or aryl;
is alkyl, aryl, heteroaryl, cycloalkyl, alkenyl, cycloalkenyl, or alkynyl;
and Ri9 are, independently, hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,
monofluoroalkyl, perfluoroalkyl, aryl, arylalkyl, cycloalkenyl, heteroaryl,
heteroarylalkyl, hydroxy-(C2-C6)alkyl, alkoxyalkyl, alkylthioalkyl, carbonyl, acyl,
alkoxycarbonyl, -C(O)NH2, alkylaminocarbonyl, dialkylaminocarbonyl, alkylaminoalkyl,
or dialkylaminoalkyl;
or Rig and Ri9 are taken together with the nitrogen atom to which they are
attached to form a saturated, unsaturated or partially saturated C4-C6 carbon ring.
In some compounds of the present invention,
, and RIO, are, independently, hydrogen, lower alkyl or
halogen;
RH, Ri2, Ri4, and Ri5 are, independently, hydrogen, hydroxy, lower alkyl, alkoxy,
or halogen;
one of and Re is hydrogen and the other is lower alkyl;
Ru is hydrogen, -(C=O)Ri6, -S(O)2Ri7, -S(O)2N(Ri8)(Ri9), or D-glucuronidate;
Ri6 is alkyl, aralkyl or aryl;
Rn is alkyl, aryl, heteroaryl, cycloalkyl, alkenyl, cycloalkenyl, or alkynyl;
RIS and Ri9 are, independently, hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,
monofluoroalkyl, perfluoroalkyl, aryl, arylalkyl, cycloalkenyl, heteroaryl,
heteroarylalkyl, hydroxy-(C2-C6)alkyl, alkoxyalkyl, alkylthioalkyl, carbonyl, acyl,
alkoxycarbonyl, -C(O)NH2, alkylaminocarbonyl, dialkylaminocarbonyl, alkylaminoalkyl,
or dialkylaminoalkyl
or Rig and Ri9 are taken together with the nitrogen atom to which they are
attached to form a saturated, unsaturated or partially saturated C4-C6 carbon ring.
The present invention also provides, inter alia, prodrugs, enantiome, hydrates,
solvates, or pharmaceutically acceptable salts or este of Formula 1.
Methods of using the compounds of the present ivnention are also provided. For
example, exemplary compounds can be used to modulate cytokine expression in a cell.
In some embodiments, an effective amount of one or more compounds of the present
invention can be provided to a subject to inhibit cytokine expression in the subject. In
one aspect, the cytokine is interleukin, e.g, IL-6. In some embodiments, the subject
suffe from chronic inflammatory disease. In some embodiments, the subject suffe
from atherosclerosis, myocardial infarction, congestive heart failure, inflammatory bowel
disease, or arthritis.
The present invention also provides, inter alia, methods of treating a condition or
disease characterized by increased cytokine expression or activity. Such methods
comprise providing to a subject a pharmaceutically effective amount of a compound of
the present invention. In one aspect, the disease is a chronic inflammatory disease. In
some embodiments, the compound is useful in treating the inflammatory component of a
disease such as atherosclerosis, myocardial infarction, congestive heart failure,
inflammatory bowel disease, or arthritis.
The present invention also provides, inter alia, methods of determining the
activity of a compound of the present invention. The method can include the step of
contacting a compound of the present invention with a cell and measuring cytokine
expression in the cell, e.g., by Western or Northern Blot. In some embodiments, the cell
will be one that overexpresses cytokines. The step of measuring cytokine expression in
the cell can occur before or after the contacting step. In one aspect, the cytokine is
interleukin, e.g., IL-6.
The present invention also provides, inter alia, kits for inhibiting cytokine
expression and/or for treating chronic inflammatory disease in a subject comprising a
container, a pharmaceutical composition contained therein comprising a compound of the
present invention, and a package insert indicating that the pharmaceutical composition
can be used for the inhibition of cytokine expression and/or for the treatment of chronic
inflammatory disease.
DETAILED DESCRIPTION
The present invention provides substituted phenanthridine carbonyl phenols and
substituted phenanthridine carbonyl phenol derivatives, processes for preparing such
compounds, pharmaceutical compositions comprising such compounds, and methods for
using such compounds. Preferred compounds have properties that are useful for the
treatment, including the prevention and inhibition, of a wide variety of diseases and
disorde affected by the estrogen receptor.
Compounds of the present invention include those of Formula 1:
Formula 1
wherein
RI, R2, , and Rj, are, independently, hydrogen, lower alkyl, halogen, or aryl;
Ry, Rg, Rg, and RIO, are, independently, hydrogen, lower alkyl or halogen;
RU, Ri2, RH, and RIS are, independently, hydrogen, hydroxy, lower alkyl, alkoxy,
or halogen;
one of and Re is hydrogen and the other is lower alkyl;
Rie is alkyl, aralkyl or aryl;
Rnis alkyl, aryl, heteroaryl, cycloalkyl, alkenyl, cycloalkenyl, or alkynyl;
Rig and Rig are, independently, hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,
monofluoroalkyl, perfluoroalkyl, aryl, arylalkyl, cycloalkenyl, heteroaryl,
heteroarylalkyl, hydroxy-(C2-Ce)alkyl, alkoxyalkyl, alkylthioalkyl, carbonyl, acyl,
alkoxycarbonyl, -C(O)NH2, alkylaminocarbonyl, dialkylaminocarbonyl, alkylaminoalkyl,
or dialkylaminoalkyl;
or Rig and Ri9 are taken together with the nitrogen atom to which they are
attached to form a saturated, unsaturated or partially saturated C4-Ce carbon ring.
In some compounds of the present invention,
RI, R2, , Rt, R Rg, R9, and RIO, are, independently, hydrogen, lower alkyl or
halogen;
RH, Ria, RM, and RIS are, independently, hydrogen, hydroxy, lower alkyl, alkoxy,
or halogen;
one of R5 and Re is hydrogen and the other is lower alkyl;
Rn is hydrogen, -(OO)Ri6, -S(0)2Ri7, -S(O)2N(Ri8)(Ri9), or D-glucuronidate;
Rie is alkyl, aralkyl or aryl;
Rn is alkyl, aryl, heteroaryl, cycloalkyl, alkenyl, cycloalkenyl, or alkynyl;
RIS and Ri9 are, independently, hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,
monofluoroalkyl, perfluoroalkyl, aryl, arylalkyl, cycloalkenyl, heteroaryl,
heteroarylalkyl, hydroxy-(C2-C6)alkyl, alkoxyalkyl, alkylthioalkyl, carbonyl, acyl,
alkoxycarbonyl, -C(O)NH2, alkylaminocarbonyl, dialkylaminocarbonyl, alkylaminoalkyl,
or dialkylaminoalkyl
or Rig and Ri9 are taken together with the nitrogen atom to which they are
attached to form a saturated, unsaturated or partially saturated CCe carbon ring.
In some compounds of the present invention
RI, R2) , and Rt, are, independently, hydrogen, Ci.6 alkyl, halogen, or Ce-n aryl;
R7, Rg, Rg, and RIO, are, independently, hydrogen, Ci-6 alkyl or halogen;
Rn, Ri2, RH, and RIS are, independently, hydrogen, hydroxy, Ci-6 alkyl, Ci.6
alkoxy, or halogen;
one of and Re is hydrogen and the other is Ci-e alkyl;
Rn is hydrogen, -(C=O)Ri6, -S(O)2Rn, -S(O)2N(Ri8)(Ri9), or D-glucuronidate;
Ri6 is Ci.6 alkyl, Ce-n ar(Ci-6)alkyl or C6-i4 aryl;
Rn is Ci-6 alkyl, Ce-n aryl, heteroaryl, Ci.6 cycloalkyl, C2.i5 alkenyl , C4.i5
cycloalkenyl, or C2-is alkynyl,
RIS and Ri9 are, independently, hydrogen, Ci.6 alkyl, C2.i5 alkenyl, C2.i5 alkynyl,
3.15 cycloalkyl, monofluoro(Ci-io)alkyl, perfluoro(Ci-6)alkyl, Ce-u aryl, Ce-n aryl(Ci.
e)alkyl, CMS cycloalkenyl, heteroaryl, heteroaryl(Ci-6)alkyl, hydroxy-(C2-C6)alkyl, C\.6
alkoxy(C].6)alkyl, Ci-e alkylthio(Ci_6)alkyls carbonyl, acyl, Ci-e alkoxycarbonyl,
C(O)NH2, Ci.6 alkylaminocarbonyl, di(Ci-6)alkylaminocarbonyl, Ci-6 alkylamino(Ci.
e)alkyl, or di(Ci.6)alkylamino(Ci-6)alkyl
or Rig and Ri9 are taken together with the nitrogen atom to which they are
attached to form a saturated, unsaturated or partially saturated C4-Ce carbon ring.
Compounds of the present invention also include prodrugs, enantiome,
diastereome, racemates, geometric isome, hydrates, solvates, or pharmaceutically
acceptable salts or este of Formula 1.
Exemplary compounds of Formula 1 include those wherein at least one of RI, Ra,
, R4, RV, Rg, Rg, and RIO, is halogen. For example, in one exemplary embodiment, RI
or Rg is halogen. In another exemplary embodiment, both and Rg are halogen. In
another exemplary embodiment, R or Rg is chlorine or fluorine or and Rg are chlorine
or fluorine. Exemplary embodiments of compounds of Formula 1 include those wherein
RI, Ra, , R4, Rj, Rg, Rg, and RIO are hydrogen or halogen.
Exemplary compounds of Formula 1 include those wherein Ri2, RH and RIS are
hydrogen, halogen, hydroxy, or alkoxy. For example in some embodiments, Ri2, RU and
RIS are hydrogen, chlorine, bromine, fluorine, hydroxy, or methoxy.
Exemplary compounds of Formula 1 include those wherein RB is hydrogen.
Exemplary embodiments of Formula 1 include those wherein at least one of RI,
Ra, , R4, R Rg, Rg, and RIO, is halogen, Ri2, RU and RIS are hydrogen, chlorine,
bromine, fluorine, hydroxy, or alkoxy (e.g., methoxy) and Rn is hydrogen.
Further exemplary embodiments of Formula 1 include those wherein at
least one of RI, R2, Ra, R4, RI, Rg, Rg, and RIO, is halogen and the othe are hydrogen.
Further embodiments include those where at least one of Ri2, RH and RIS is hydrogen,
chlorine, bromine, fluorine, hydroxy, or alkoxy (e.g., methoxy) and the othe are
hydrogen. For both of these embodiments Rn is preferably hydrogen.
Representative compounds of the present invention include compounds of
wherein RI, R2, , R4, , Re RV, , RQ, RIO, RII, Ri2, RH, and Ri5 are as defined
herein for Formula 1 .
The present invention also provides, inter alia, prodrugs, enantiome,
diastereome, racemates, geometric isome, hydrates, solvates, or pharmaceutically
acceptable salts or este of compounds of Formulas 2 to 12.
Compounds of Formulas 2 to 12 include those wherein at least one of RI, R2, ,
RI, RT, Rg, R9, and RIO, is halogen. For example, in one exemplary embodiment, or Rg
is halogen. In another exemplary embodiment, both or Rg are halogen. In another
exemplary embodiment, or Rg is chlorine or fluorine or and Rg are chlorine or
fluorine. Exemplary compounds of Formulas 2 to 12 include those wherein at RI, R2,
, R4, R Rg, Rg, and RIO are hydrogen or halogen.
Exemplary compounds of Formulas 2 to 12 include those wherein Ri2, RH and
RIS are hydrogen, halogen, hydroxy, or alkoxy. For example in some embodiments, R,
RH and RIS are hydrogen, chlorine, bromine, fluorine, hydroxy, or methoxy.
Exemplary embodiments of Formulas 2 to 12 include those wherein at least one
of RI, R2, , R4, R Rg, Rg, and RIO, is halogen and Rn, RH and RIS are hydrogen,
chlorine, bromine, fluorine, hydroxy, or methoxy.
Exemplary substituted 6-substituted-phenanthridin-5(6//)-ylcarbonylphenyl
derivatives of the present invention include, but are not limited to, 4-{[(6/?)-8-fluoro-6-
methylphenanthridin-5(6//)-yl]carb°nyl}phen°U 3-{[(65)-8-fluoro-6-
methylphenanthridin-5(6//)-yl]carbonyl}phenol;4-{[(65)-8-fluoro-6-
methylphenanthridin-5(6/f)-yl]carbonyl}phenol;3-{[(6)-8-fluoro-6-
methylphenanthridin-5(6//)-yl]carbonyl}phenol; 3-fluoro-4-{[(6/?)-8-fluoro-6-
methylphenanthridin-5(6//)-yl]carbonyl}phenol; 3-fluoro-4-{[(65)-8-fluoro-6-
methylphenanthridin-5(6/f)-yl]carbonyl}phenol;4-fluoro-3-{[(6)-8-fluoro-6-
methylphenanthridin-5(6//)-yl]carbonyl}phenol;4-fluoro-3-{[(65)-8-fluoro-6-
methylphenanthridin-5(6//)-yl]carbonyl}phenol;2-fluoro-4-{[(6)-8-fluoro-6-
methylphenanthridin-5(6//)-yl]carbonyl}phenol;2-fluoro-4-{[(65<)-8-fluoro-6-
methylphenanthridin-5(6//)-yl]carbonyl}phenol; 2-chloro-5-{[(6/?)-8-fluoro-6-
methylphenanthridin-5(6//)-yl]carbonyl}phenol; 2-chloro-5-{[(65)-8-fluoro-6-
methylphenanthridin-5(6/f)-yl]carbonyl}phenol; 2-bromo-4-{[(6/?)-8-fluoro-6-
methylphenanthridin-5(6/T)-yl]carbonyl}phenol; 2-bromo-4-{[(6S)-8-fluoro-6-
methylphenanthridin-5(6//)-yl]carbonyl}phenol;4-bromo-3-{[(6/?)-8-fluoro-6-
methylphenanthridin-5(6//)-yl]carbonyl}phenol;4-bromo-3-{[(6S)-8-fluoro-6-
methylphenanthridin-5(6//)-yl]carbonyl}phenol;4-{[(6/?)-8-fluoro-6-
methylphenanthridin-5(6/f)-yl]carbonyl}-2-methoxyphenol; 4-{[(65)-8-fluoro-6-
methylphenanthridin-5(6//)-yl]carbonyl}-2-methoxyphenol; 4-{[(6S)-6-ethyl-8-
fluorophenanthridin-5(6//)-yl]carbonyl}phenol;4-{[(6/?)-6-ethyl-8-fluorophenanthridin-
5(6/f)-yl]carbonyl}phenol;3-{[(6)-6-ethyl-8-fluorophenanthridin-5(6//)-
yl]carbonyl}phenol; 4-{[(6/J)-6-ethyl-8-fluorophenanthridin-5(6//)-yl]carbonyl}benzene-
1,3-diol; 4-[(6-ethyl-8-fluorophenanthridin-5(6/f)-yl) carbonyl]-3-fluorophenol; 3-[(3-
chloro-6-methylphenanthridin-5(6//)-yl)carbonyl]phenol; 3- {[(6J?)-3-chloro-6-
methylphenanthridin-5(6Jc/)-yl]carbonyl}phenol; 3-{[(6S)-3-chloro-6-
methylphenanthridin-5(6//)-yl]carbonyl}phenol;4-[(3-chloro-6-methylphenanthridin-
5(6/f)-yl)carbonyl]phenol; 4-{[(6/?)-3-chloro-6-methylphenanthridin-5(6//)-
yl]carbonyl}phenol; 4-{ [(65)-3-chloro-6-methylphenanthridin-5(6//)-
yl] carbonyl} phenol;
4-[3-chloro-6-methylphenanthridin-5(6/f)-yl]carbonyl]benzene-l,3-diol;4-{[(6/?)-3-
chloro-6-methylphenanthridin- 5 (6H)-y\] carbonyl} benzene-1,3 -diol; 4- {[(65)- 3-chloro-6-
methylphenanthridin-5(6//)-yl]carbonyl}benzene-1,3-diol; 3-[(3-fluoro-6-
methylphenanthridin-5(6//)-yl)carbonyl]phenol; 3-{[(6/?)-3-fluoro-6-
methylphenanthridin-5(6//)-yl]carbonyl}phenol;3-{[(65)-3-fluoro-6-
methylphenanthridin-5(6/)-yl]carbonyl}phenol;4-[(3-fluoro-6-methylphenanthridin-
5(6#)-yl)carbonyl]phenol; 4- {[(6/?)-3-fluoro-6-methylphenanthridin-5(6//)-
yl]carbonyl}phenol;4-{[(6S)-3-fluoro-6-methylphenanthridin-5(6//)-yl]carbonyl}phenol;
4-[(3-fluoro-6-methylphenanthridin-5(6//)-yl)carbonyl]benzene-l,3-diol;4-{[(6/?)-3-
fluoro-6-methylphenanthridin-5(6//)-yl]carbonyl} benzene-1,3-diol; 4-{ [(6S)-3-fluoro-6-
methylphenanthridin-5(6//)-yl]carbonyl}benzene-l,3-diol;4-[(3,8-difluoro-6-
methylphenanthridin-5(6//)-yl)carbonyl]phenol;4-{[(6)-3,8-Difluoro-6-
methylphenanthridin-5(6//)-yl]carbonyl}phenol; 4-{[(6S)-3,8-Difluoro-6-
methylphenanthridin-5(6/)-yl]carbonyl}phenol and pharmaceutically acceptable salt and
ester forms thereof.
As used herein alone or as part of a group, the term "alkyl" includes both
branched and straight-chain saturated aliphatic hydrocarbon groups having the specified
number of carbon atoms, e.g. methyl (Me), ethyl (Et), propyl (Pr), isopropyl (i-Pr),
isobutyl (i-Bu), secbutyl (s-Bu), tertbutyl (t-Bu), isopentyl, isohexyl and the like. The
term "alkyl" further includes both unsubstituted and mono-, di- and tri-substituted
hydrocarbon groups, suitable substituents are selected from acyloxy, hydroxy, acyl, alkyl
of 1 to 6 carbon atoms, alkoxy of 1 to 6 carbon atoms, alkenyl of 2 to 6 carbon atoms,
alkynyl of 2 to 6 carbon atoms, amino, amino substituted by one or two alkyl groups of
from 1 to 6 carbon atoms, aminoacyl, acylamino, azido, cyano, halo, nitro, thioalkoxy of
from 1 to 6 carbon atoms, thioalkoxy of from 1 to 6 carbon atoms mono- or di-substituted
with lower alkyl or alkoxy, trihalomethyl, aryl, and heteroaryl, with halogen substitution
particularly preferred. Preferred alkyl groups have from 1 to 12 carbon atoms, more
preferably from 1 to 6 carbon atoms unless otherwise defined. A lower alkyl has from 1
to 6 carbon atoms.
The carbon number as used in the definitions herein refe to carbon backbone
and carbon branching, but does not include carbon atoms of the substituents, such as
alkoxy substitutions and the like.
The term "alkenyl" as used herein alone or as part of a group refe to an
unsaturated or partially unsaturated aliphatic hydrocarbon group having the specified
number of carbon atoms, for example ethenyl, 1-propenyl, 2, butenyl, etc. The term
13
"alkenyl" further includes both unsubstituted and mono-, di- and tri-substituted
hydrocarbon groups, suitable substituents are selected from acyloxy, hydroxy, acyl, alkyl
of 1 to 6 carbon atoms, alkoxy of 1 to 6 carbon atoms, alkenyl of 2 to 6 carbon atoms,
alkynyl of 2 to 6 carbon atoms, amino, amino substituted by one or two alkyl groups of
from 1 to 6 carbon atoms, aminoacyl, acylamino, azido, cyano, halo, nitro, thioalkoxy of
from 1 to 6 carbon atoms, thioalkoxy of from 1 to 6 carbon atoms mono- or di-substituted
with lower alkyl or alkoxy, trihalomethyl, aryl, and heteroaryl, with halogen substitution
particularly preferred. Preferred alkenyl groups have from 1 to 12 carbon atoms.
The term "alkynyl", as used herein alone or as part of a group refe to a
substituted or unsubstituted aliphatic hydrocarbon chain and includes, but is not limited
to, straight and branched chains and containing at least one triple bond. Preferably, the
alkynyl moiety has 2 to 10 carbon atoms. In certain embodiments, the alkynyl can
contain more than one triple bond and, in such cases, the alknyl group must contain at
least three carbon atoms. Specifically included within the definition of "alkynyl" are
those aliphatic hydrocarbon chains that are optionally substituted. Suitable substituents
are selected from acyloxy, hydroxy, acyl, alkyl of 1 to 6 carbon atoms, alkoxy of 1 to 6
carbon atoms, alkenyl of 2 to 6 carbon atoms, alkynyl of 2 to 6 carbon atoms, amino,
amino substituted by one or two alkyl groups of from 1 to 6 carbon atoms, aminoacyl,
acylamino, azido, cyano, halo, nitro, thioalkoxy of from 1 to 6 carbon atoms, thioalkoxy
of from 1 to 6 carbon atoms mono- or di-substituted with lower alkyl or alkoxy,
trihalomethyl, aryl, and heteroaryl.
The term "perfluoroalkyl", as used herein, whether used alone or as part of
another group, refe to an alkyl group substituted with two or more fluorine atoms and
includes, but is not limited to, straight or branched chains, such as -CF, -CFCFs,
-CF2CF3 and -CH(CF3)2.
The term "carbonyl" as used herein alone or as part of a group refe to the group
The term "acyl", employed alone or in combination with other terms, is defined
herein as, unless otherwise stated, either an arylalkyl, heteroarylalkyl, (Ca-Cio) straight
chain, or (C4-Cu) branched-chain monovalent hydrocarbon moiety; wherein the carbon
atom, covalently linked to the defined chemical structure, is oxidized to the carbonyl
oxidation state. Such hydrocarbon moieties may be mono or polyunsaturated, and may
exist in the E or Z configurations. The compounds of this invention are meant to include
all possible E and Z configurations. Examples of acyl moieties include, but are not
limited to, chemical groups such as acetyl, propionyl, butyryl, 3,3-dimethylbutyryl,
trifluoroacetyl, pivaloyl, hexanoyl, hexenoyl, decanoyl, benzoyl, nicotinyl, isonicotinyl,
and homologs, isome, and the like.
The term "alkylamino" as used herein alone or as part of a group refe to the
group -NH-alkyl
The term "alkylaminocarbonyl" as used herein alone or as part of a group refe to
an alkylamino group bonded through a carbonyl group.
The term "dialkylamino" herein alone or as part of a group refe to the group -
N(alkyl)2, where the alkyl group is the same or different.
The term "dialkylaminocarbonyl" as used herein alone or as part of a
group refe to a dialkylamino group bonded through a carbonyl group.
The term "alkylaminocarbonyl" as used herein alone or as part of a group refe to
an alkylamino group bonded through a carbonyl group.
The term "alkoxy" as used herein alone or as part of a group refe to the
group Ra-O- wherein Ra is an alkyl group as defined above.
The term" alkoxycarbonyl" as used herein alone or as part of a group
refe to an alkoxy group bonded through a carbonyl group.
The term "cycloalkyl" includes cyclized alkyl chains having the specified
number of carbon atoms, e.g., cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.
The term "cycloalkenyl" includes cyclized alkyl chains containing an
alkenyl group having the specified number of carbon atoms, e.g., cyclopentenyl,
cyclohexenyl, and the like. Specifically included within the definition of cyclolalkyl and
cycloalkenyl are those cycloalkyl groups that are optionally substituted. Suitable
substituents are selected from acyloxy, hydroxy, acyl, alkyl of 1 to 6 carbon atoms,
alkoxy of 1 to 6 carbon atoms, alkenyl of 2 to 6 carbon atoms, alkynyl of 2 to 6 carbon
atoms, amino, amino substituted by one or two alkyl groups of from 1 to 6 carbon atoms,
aminoacyl, acylamino, azido, cyano, halo, nitro, thioalkoxy of from 1 to 6 carbon atoms,
thioalkoxy of from 1 to 6 carbon atoms mono- or di-substituted with lower alkyl or
alkoxy, trihalomethyl, aryl, and heteroaryl. Preferred cycloalkyl and cycloalkenyl groups
contain from 3 to 15 carbon atoms.
The term "halogen" includes fluorine, chlorine, iodine, and bromine.
The term "aryl" refe to an aromatic carbocyclic moiety of up to 20 carbon
atoms, which can be a single ring (monocyclic) or multiple rings (bicyclic, up to three
rings) fused together or linked covalently. Any suitable ring position of the aryl moiety
can be covalently linked to the defined chemical structure. Examples of aryl moieties
include, but are not limited to, chemical groups such as phenyl, 1-naphthyl, 2-naphthyl,
dihydronaphthyl, tetrahydronaphthyl, biphenyl, anthryl, phenanthryl, fluorenyl, indanyl,
biphenylenyl, acenaphthenyl, acenaphthylenyl, and the like. Specifically included with
the term "aryl" are those aryl groups that are optionally substituted. Suitable substituents
are selected from acyloxy, hydroxy, acyl, alkyl of 1 to 6 carbon atoms, alkoxy of 1 to 6
carbon atoms, alkenyl of 2 to 6 carbon atoms, alkynyl of 2 to 6 carbon atoms, amino,
amino substituted by one or two alkyl groups of from 1 to 6 carbon atoms, aminoacyl,
acylamino, azido, cyano, halo, nitro, thioalkoxy of from 1 to 6 carbon atoms, thioalkoxy
of from 1 to 6 carbon atoms mono- or di-substituted with lower alkyl or alkoxy,
trihalomethyl, aryl, and heteroaryl. Preferred aryl groups contain from 6 to 14 carbon
atoms.
The term "arylalkyl", as used herein, whether used alone or as part of another
group, refe to the group -Ra-Rb, where Ra is an alkyl group as defined above, substituted
by Rb, an aryl group, as defined above. In a preferred embodiment, the alkyl chain is
either a (Q-Ce) straight or (2-07) branched-chain saturated hydrocarbon moiety.
Examples of arylalkyl moieties include, but are not limited to, chemical groups such as
benzyl, 1-phenyl ethyl, 2-phenylethyl, diphenylmethyl, 3-phenylpropyl, 2-phenylpropyl,
fluorenylmethyl, and homologs, isome, and the like. These may be optionally
substituted as discussed for the aryl and alkyl groups defined above.
The term "heterocyclic ring or ring system", employed alone or in combination
with other terms, is defined herein as an unsaturated, partially unsaturated or saturated
ring or ring system, which can be a single ring (monocyclic) or multiple rings (bicyclic,
up to three rings) fused together or linked covalently. The rings can contain from one to
four hetero atoms selected from nitrogen (N), oxygen (O), or sulfur (S), wherein the
nitrogen or sulfur atom(s) are optionally oxidized, or the nitrogen atom(s) are optionally
quarternized. Any suitable ring position of the heteroaryl moiety can be covalently
linked to the defined chemical structure. Examples of unsaturated heterocyclic rings or
ring systems include, but are not limited to, heterocycles such as furan, thiophene,
pyrrole, N-methylpyrrole, pyrazole, N-methylpyrazole, imidazole, N-methylimidazole,
oxazole, isoxazole, thiazole, isothiazole, IH-tetrazole, 1-methyltetrazole, 1,3,4-
oxadiazole, lH-l,2,4-triazole, 1 -methyl-1,2,4-triazole 1,3,4-triazole, 1-methyl-1,3,4-
triazole, pyridine, pyrimidine, pyrazine, pyridazine, benzoxazole, benzisoxazole,
benzothiazole, benzofuran, benzothiophene, thianthrene, dibenzo[b,d]furan,
dibenzo[b,d]thiophene, benzimidazole, N-methylbenzimidazole, indole, indazole,
quinoline, isoquinoline, quinazoline, quinoxaline, purine, pteridine, 9H-carbazole, Pcarboline,
and the like. Examples of saturated or partially unsaturated heterocyclic rings
or ring systems include, but are not limited to, chemical groups such as azetidinyl, 1,4-
dioxanyl, hexahydroazepinyl, piperazinyl, piperidinyl, pyrrolidinyl, morpholinyl,
thiomorpholinyl, dihydrobenzimidazolyl, dihydrobenzofuranyl, dihydrobenzothienyl,
dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl,
dihydroisooxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl,
dihydropyrrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl,
dihydropyrrolyl, dihydroquinolinyl, dihydrotetrazolyl, dihydrothiadiazolyl,
dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, dihydro-1,4-
dioxanyl, tetrahydrofuranyl, tetrahydrothienyl, tetrahydroquinolinyl,
tetrahydroisoquinolinyl, and the like. Specifically included within the term "heterocycle"
are those heterocyclic groups that are optionally substituted. Suitable substituents are
selected from acyloxy, hydroxy, acyl, alkyl of 1 to 6 carbon atoms, alkoxy of 1 to 6
carbon atoms, alkenyl of 2 to 6 carbon atoms, alkynyl of 2 to 6 carbon atoms, amino,
amino substituted by one or two alkyl groups of from 1 to 6 carbon atoms, aminoacyl,
acylamino, azido, cyano, halo, nitro, thioalkoxy of from 1 to 6 carbon atoms, thioalkoxy
of from 1 to 6 carbon atoms mono- or di-substituted with lower alkyl or alkoxy, and
trihalomethyl.
As used herein, the term "heteroaryl", whether used alone or as part of another
group, is defined as a substituted or unsubstituted aromatic heterocyclic ring system
(monocyclic or bicyclic). Heteroaryl groups can have, for example, from about 3 to
about 50 carbon atoms (unless explicitly specified otherwise) with from about 4 to about
10 being preferred. In some embodiments, heteroaryl groups are aromatic heterocyclic
rings systems having about 4 to about 14 ring atoms and containing carbon atoms and 1,
2, 3, or 4 heteroatoms selected from oxygen, nitrogen or sulfur. Representative
heteroaryl groups are furan, thiophene, indole, azaindole, oxazole, thiazole, isoxazole,
isothiazole, imidazole, N-methylimidazole, pyridine, pyrimidine, pyrazine, pyrrole, Nmethylpyrrole,
pyrazole, N-methylpyrazole, 1,3,4-oxadiazole, 1,2,4-triazole, 1-methyl-
1,2,4-triazole, IH-tetrazole, 1-methyltetrazole, benzoxazole, benzothiazole, benzofuran,
benzisoxazole, benzimidazole, N-methylbenzimidazole, azabenzimidazole, indazole,
quinazoline, quinoline, and isoquinoline. Bicyclic aromatic heteroaryl goups include
phenyl, pyridine, pyrimidine or pyridizine rings that are (a) fused to a 6-membered
aromatic (unsaturated) heterocyclic ring having one nitrogen atom; (b) fused to a 5- or 6-
membered aromatic (unsaturated) heterocyclic ring having two nitrogen atoms; (c) fused
to a 5-membered aromatic (unsaturated) heterocyclic ring having one nitrogen atom
together with either one oxygen or one sulfur atom; or (d) fused to a 5-membered
aromatic (unsaturated) heterocyclic ring having one heteroatom selected from O, N or S.
Specifically included within the definition of "heteroaryl" are those aromatic heterocyclic
rings that are substituted, for example with 1 to 5 substituents selected from the group
consisting of acyloxy, hydroxy, acyl, alkyl of 1 to 6 carbon atoms, alkoxy of 1 to 6
carbon atoms, alkenyl of 2 to 6 carbon atoms, alkynyl of 2 to 6 carbon atoms, amino,
amino substituted by one or two alkyl groups of from 1 to 6 carbon atoms, aminoacyl,
acylamino, azido, cyano, halo, nitro, thioalkoxy of from 1 to 6 carbon atoms, thioalkoxy
of from 1 to 6 carbon atoms mono- or di-substituted with lower alkyl or alkoxy, and
trihalomethyl. Where there is more than one substituent the substituents may be the same
or different.
The compounds of the present invention can be converted to salts, in particular
pharmaceutically acceptable salts using art recognized procedures. The compounds of
formulas 1 to 12 that have a basic center can form acid addition salts. These are formed,
for example, with strong inorganic acids, such as mineral acids for example sulfuric acid,
phosphoric acid or a hydrohalic acid, with strong organic carboxylic acids, such as
alkanecarboxylic acids of 1 to 4 carbon atoms which are unsubstituted or substituted, for
example, by halogen, for example acetic acid such as saturated or unsaturated
dicarboxylic acids, for example oxalic, malonic, succinic, maleic, fumaric, phthalic, or
terephthalic acid, such as hydroxycarboxylic acids, for example ascorbic, glycolic, lactic,
malic, tartaric or citric acid, such as amino acids, for example aspartic or glutamic acid,
or such as benzoic acid, or with organic sulfonic acids, such as alkane- (of 1 to 4 carbon
atoms) or arylsulfonic acids, for example methane- or p-toluenesulfonic acid.
Corresponding acid addition salts can also be formed having, if desired, an additionally
present basic center. The compounds of formulas 1 to 12 having at least one acid group
can form salts with bases. Suitable salts with bases are, for example, metal salts, such as
alkali metal or alkaline earth metal salts, for example sodium, potassium or magnesium
salts, or salts with ammonia or an organic amine, such as morpholine, thiomorpholine,
piperidine, pyrrolidine, a mono-, di- or tri-lower alkylamine, for example ethyl-tert-butyl-
, diethyl-, diisopropyl-, triethyl-, tributyl- or dimethylpropylamine, or a mono-, di-, or
trihydroxy lower alkylamine, for example mono-, di- or triethanolamine. Internal salts
can furthermore be formed. Salts which are unsuitable for pharmaceutical uses but which
can be employed, for example, for the isolation or purification of free compounds of
formulas 1 to 12 or their pharmaceutically acceptable salts, are also included.
Pharmaceutically acceptable este refe to este that are pharmaceutically
acceptable and have the desired pharmacological properties. Pharmaceutically acceptable
este include este formed from carboxy, sulfonyloxy, and phosphonoxy groups present
in the compounds, e.g. C\ alkyl este. When there are two acidic groups present, a
pharmaceutically acceptable ester can be a mono-acid-mono- ester or a di-ester; and
similarly where there are more than two acidic groups present, some or all of such groups
can be esterified.
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 effective amount of the compound or substance within the
body.
Certain of the compounds of Formulas 1 to 12 contain stereogenic carbon atoms
or other chiral elements and thus give rise to stereoisome, including enantiome and
diastereome. All stereoisome of the compounds of the instant invention are
contemplated, either in admixture or in pure or substantially pure form. The definition of
the compounds according to the invention embraces all possible stereoisome and their
mixtures. Throughout this application, the name of the product, where the absolute
configuration of an asymmetric center is not indicated is intended to embrace the
individual stereoisome as well as mixtures of stereoisome.
Where an enantiomer (or a particular blend of enantiome) is preferred, it can, in
some embodiments, be provided substantially free of the corresponding enantiomer(s).
Thus, an enantiomer substantially free of the corresponding enantiomer refe to a
compound that is isolated or separated via separation techniques or prepared free of the
corresponding enantiomer. "Substantially free," as used herein, means that the
compound is made up of a significantly greater proportion of one enantiomer. In
preferred embodiments, the compound is made up of at least about 90% by weight of a
preferred enantiomer. In other embodiments of the invention, the compound is made up
of at least about 99% by weight of a preferred enantiomer. Preferred enantiome can be
isolated from racemic mixtures by any method known to those skilled in the art,
including high performance liquid chromatography (HPLC) and the formation and
crystallization of chiral salts, or preferred enantiome can be prepared by methods
described herein. The racemic forms can be resolved by physical methods, such as, for
example, fractional crystallization, separation or crystallization of diastereomeric
derivatives or separation by chiral column chromatography. The individual optical
isome can be obtained from the racemates by conventional methods, such as, for
example, salt formation with an optically active acid followed by crystallization.
Methods for the preparation of preferred enantiome are described, for example, in
Jacques, et al., Enantiome, Racemates and Resolutions (Wiley Intecience, New York,
1981); Wilen, S.H., et al., Tetrahedron 33:2725 (1977); Eliel, E.L. Stereochemistry of
Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, S.H. Tables of Resolving
Agents and Optical Resolutions p. 268 (E.L. Eliel, Ed., Univ. of Notre Dame Press, Notre
Dame, IN 1972), each of which is incorporated herein by reference in its entirety and for
all purposes. Accordingly, the present invention embraces the racemic forms of the
claimed compounds and the isolated optical isome having the specified activity.
It should be undetood that the present invention includes prodrug forms of the
compounds of Formulas 1 to 12. Various forms of prodrugs are well known in the art.
For examples of such prodrug derivatives, see: (a) Design of Prodrugs, edited by H.
Bundgaard (Elsevier, 1985); and Methods in Enzymology, Vol. 42, pp. 309-396, edited
by K.Widder et al., (Academic Press, 1985); (b) A Textbook of Drug Design and
Development, edited by Krosgaard-Laen and H. Bundgaard, Chapter 5, "Design and
Application of Prodrugs," by H. Bundgaard, pp. 113-191 (1991); (c) H. Bundgaard,
Advanced Drug Deliver Reviews, 8, pp. 1-38 (1992); (d) H. Bundgaard et al., Journal of
Pharmaceutical Sciences, 77, 285 (1988); and (e) N. Kayeka et al., Chem. Phar. Bull., 32,
692 (1984), each of which is incorporated herein by reference in its entirety and for all
purposes. For example, a representative scheme for synthesizing an exemplary prodrug of
the present invention is as follows:
R»
RIO
RCOC1
DIEA, CH2Cl2
OH
Solvates (e.g., hydrates) are also within the scope of the present invention.
Methods of solvation are generally known in the art. Accordingly, the compounds of the
instant invention can be in the free or hydrate form, and can be obtained by methods
exemplified by the following schemes below.
Compounds of the present invention can be prepared by those skilled in the art of
organic synthesis employing conventional methods that utilize readily available reagents
and starting materials. The compounds of the present invention can be prepared from
commercially available starting materials, compounds known in the literature, or readily
prepared intermediates, by employing standard synthetic methods and procedures known
to those skilled in the art. Standard synthetic methods and procedures for the preparation
of organic molecules and functional group transformations and manipulations can be
readily obtained from the relevant scientific literature or from standard textbooks in the
field. Although not limited to any one or several sources, classic texts such as Smith, M.
B.; March, J. March's Advanced Organic Chemistry: Reactions, Mechanisms, and
Structure, 5th ed.; John Wiley & Sons: New York, 2001; and Greene, T. W.; Wuts, P. G.
M. Protective Groups in Organic Synthesis, 3rd ed.; John Wiley & Sons: New York, 1999
are useful and recognized reference textbooks of organic synthesis known to those in the
art. The following synthetic schemes are designed to illustrate, but not limit, general
procedures for the preparation of representative compounds of the present invention. The
skilled practitioner will know how to make use of variants of these process steps.
In scheme I the compounds can be conveniently prepared from an appropriately
substituted phenanthridine. A general preparation of the phenanthridines is described by
M. Lysen, J. L. Kristensen, M. Begtrup; Organic Lette, 2002, 4, 257. Several other
methods of preparation of substituted phenanthridines are known in the literature (see P
K. Patra, J. R. Suresh, H. Ila, H. Junjappa, Tetrahedron, 1998, 54, 10167). In scheme I,
step a, a suitably substituted phenanthridine (1), either commercially available, known in
the literature, or prepared according to methods known and established for the
preparation of said phenanthridines, including the schemes described below; wherein, RI
through RIS are herein before defined, is reacted with an -Li reagent, either
commercially available, known in the literature, or prepared according to methods known
and established for the formation of lithium reagents, in a suitable solvent, such as diethyl
ether, tetrahydrofuran, 1,4-dioxane, or the like, at temperatures between -78 °C and room
temperature. The lithium amide salt (2A) is reacted in situ with a suitably substituted
benzoyl chloride (3), either commercially available, known in the literature, or prepared
according to methods known and established for the preparation of said benzoyl
chlorides. Alternatively, the suitably substituted phenanthridine is reduced in situ to the
intermediate dihydrophenanthridine (2B) in the presence of a suitable benzoyl chloride
(3), as previously described, in a non-reactive solvent, such as diethyl ether,
tetrahydrofuran, and the like, at temperatures between -78 °C and room temperature to
provide the protected phenol (4). A methyl ether (4); wherein, R\i is a methyl group, can
be de-methylated in step d to the corresponding phenol (5) by reacting (4) with boron
tribromide in the presence of an excess of olefin or cycloolefin, such as cyclohexene,
acting as a bromine and hydrogen bromide scavenger, in a suitably halogenated solvent
such as chloroform, 1,2-dichloroethane, or dichloromethane, and the like. Alternatively,
the methyl ether (4) can be de-methylated to phenol (5) by reacting (4) with boron
trichloride, in the presence of quaternary ammonium iodides, such as
tetrabutylammonium iodide, in a suitably halogenated solvent, such as chloroform, 1,2-
dichloroethane, or dichloromethane, and the like, at temperatures between -78 °C and
room temperature for two to twenty-four hou.
If RIS is a benzyl or diphenylmethyl protecting group, removal by suitably
compatible hydrogenolysis techniques, known in the literature for effecting such
transformations, such as hydrogen and 5% palladium-on-carbon catalysts will afford the
desired phenol (5).
If -ORis is a carbonate moiety, treatment of (4) with a 1 N sodium hydroxide
solution in methanol at typically 40 °C to 75 °C, for a length of time, typically about
twelve hou, will remove the protecting group to afford the desired phenol (5).
23
Scheme I
a. R5Li, ether solvent, -78 °C to RT; b. (3) at -78 °C to RT; c. (3), DIPEA, THF; or
when R5=H (CH3)2-BH3, (S)-2-methyl-CBS-oxazaborolidine, THF d. When R13 is
methyl: BB, cyclohexene, methylene chloride, 2 - 24h., (when R =H, preparative
chiral HPLC resolution)
In Scheme II, step aa, a suitably substituted arylboronic acid or ester (6), either
commercially available, known in the literature, or prepared according to methods known
and established for the preparation of said arylboronic acids or este, including
procedures exemplified in the experimental section of this application; wherein, L is a
fluorine or chlorine atom, RI through RIS, are herein before defined and RIO and Rai can
be hydrogen, lower alkyl or bridged alkyl, in the presence of a coupling catalyst, is
reacted with a suitably substituted alkyl aryl ketone (7), either commercially available,
known in the literature, or prepared according to methods known and established for the
preparation of said alkyl aryl ketones, including procedures exemplified in the
experimental section of this application; wherein, W and Re through RIO are herein before
defined. The biphenylketone (8) is reacted in step bb with an ammonium source, such as
ammonium chloride or ammonium acetate, and the like, either commercially available, or
known in the literature, in a suitable solvent such as methanol, toluene, tetrahydrofuran,
1,2-dichloroethane, and the like, optionally in the presence of an acid catalyst such as ptoluenesulfonic
acid or pyridinium /?-toluenesulfonate, followed in a second step cc by
reduction of the intermediate imine with an acceptable hydride source, such as sodium
cyanoborohydride, sodium borohydride, lithium aluminum hydride, or
diisobutylaluminum hydride, or the like. The intermediate imine can be isolated or not
isolated. The conjugate base of a suitable, nucleophilic , such as methyllithium, tertbutyllithium,
and the like, can be substituted for the hydride source to afford a tertiary
biphenylamine as the product of step cc. The biphenylamine (9) can be separated into it's
respective enantiome (95) and (9R) by subjecting (9) to either an analytical chiral
separation, an enzymatic or chemical resolution, or a de novo enantiospecific synthesis of
either (95) or (9/f) according to methods known and established in the literature for the
enantiospecific synthesis of benzylic amines. The chemical resolution of (9) is carried
out by employing a suitable chiral acid, either commercially available or known in the
literature, according to methods known and established for the resolution of benzylic
amines. The biphenylamine (9), either as the racemate or enantiomerically pure, is then
reacted in step dd with an acid chloride or anhydride, in a suitable solvent such as
acetonitrile, 1,2-dichloroethane, or dichloromethane, and the like, optionally in the
presence of an acid scavenger such as triethylamine, diisopropylethylamine, pyridine, or
potassium carbonate, and the like, and further optionally in the presence of a known
acylation promoter or catalyst, such as 4-(W)Af-dimethylamino)pyridine at -20 °C to room
temperature for several hou. In step ee, treatment of the carboxamideamide (10) with
lithium or potassium hexamethyldisilamide, LDA and the like, either commercially
available, or known in the literature in a suitable solvent such as tetrahydrofuran at 70 °C
for 24 - 48 hou afforded phenanthridine (4). The protected phenol can be de-protected
in step ff to phenol (5) as previously described.
aa. Pd(OAc)2, K2CO3, tetrabutylammonium bromide, THF, 60 °C, 2 - 12h; bb.
NH4OAc, MeOH, 60 °C; cc. When R5 is hydrogen, NaCNBH3, MeOH, 60 °C, 12h; dd.
ArCOCl wherein Rn is methyl: triethylamine, CH2C12, 2 - 12h; ee. KHMDS, THF, 70
°C, 16h; ff. When Ri3 is methyl: BBr3, cyclohexene, dichloromethane, 2 - 24h.
In preferred embodiments, the present invention concerns methods of treating a
subject comprising the step of providing a therapeutically effective amount of a ligand
which modulates NF-kB transcription factor by interaction with estrogen receptor ER-a,
estrogen receptor ER-p, or both ER-a and ER-P estrogen recepto, preferably with a
substantial absence of creatine kinase stimulation. In certain preferred embodiments, the
administration is with a substantial absence of uterotropic activity. Preferred compounds
of the present invention modulate NF-kB transcription factor by interaction with estrogen
receptor ER-a, estrogen receptor ER-P, or both ER-a and ER-p estrogen recepto
preferably with a substantial absence of creatine kinase stimulation.
Preferred compounds of the present invention modulate, e.g., inhibit cytokine
expression. For example, preferred compounds inhibit NF-kB activity and thus IL-6
expression. Accordingly, preferred compounds of the present invention possess antiinflammatory
activity and are anti-inflammatory compounds useful for the treatment of
diseases associated with increased cytokine, e.g., IL-6, expression, such as chronic
inflammatory disease. In a particularly preferred embodiment, compounds of the present
invention do not induce creatine kinase expression and accordingly, unlike classic
estrogens, do not stimulate uterine and breast cell proliferation.
The compounds according to the present invention have pharmacological
properties that can be confirmed by a number of pharmacological assays, including the
IL-6 and creatine kinase assays described in the example section herein. Preferably the
compounds of the present invention can be used to treat chronic inflammatory disease
such as atherosclerosis, myocardial infarction (MI), congestive heart failure (CHF),
inflammatory bowel disease, e.g., ulcerative colitis and Crohn's disease, and arthritis,
e.g., rheumatoid arthritis, and other diseases and conditions including those disclosed
herein. For the purposes of the present invention, a chronic inflammatory disease is any
disease characterized by chronic inflammation.
Preferred compounds of this invention are useful in treating osteoporosis and in
the inhibition of bone demineralization, which may result from an imbalance in an
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
28
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,
preferred 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.
Preferred compounds of the present invention are also active in the brain and are
therefore useful for inhibiting or treating Alzheimer's disease, cognitive decline,
decreased libido, senile dementia, neurodegenerative disorde, depression, anxiety,
insomnia, schizophrenia, and infertility. Preferred compounds of the present invention
are also useful in treating benign or malignant abnormal tissue growth including,
glomerulosclerosis, prostatic hypertrophy, uterine leiomyomas, breast cancer,
scleroderma, fibromatosis, endometriosis, endometrial cancer, polycystic ovary
syndrome, endometrial polyps, benign breast disease, adenomyosis, ovarian cancer,
melanoma, prostate cancer, cance of the colon, CNS cance, such as glioma or
astioblastomia.
Preferred compounds of the present invention are cardioprotective and are
antioxidants, and 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.
Preferred compounds of the present invention are also useful in treating disorde
associated with inflammation or autoimmune diseases, including inflammatory bowel
disease (Crohn's disease, ulcerative colitis, indeterminate colitis), arthritis (rheumatoid
arthritis, spondyloarthropathies, osteoarthritis), pleurisy, ischemia/reperfusion injury
(e.g., stroke, transplant rejection, myocardial infarction, etc.), asthma, giant cell arteritis,
prostatitis, uveitis, psoriasis, multiple sclerosis, systemic lupus erythematosus and sepsis.
Preferred compounds of the present invention are also useful in treating ocular
disorde including cataracts, uveitis, and macular degeneration and in treating skin
conditions such as aging, alopecia, and acne.
Preferred compounds of the present invention are also useful in treating metabolic
disorde such as type-II diabetes, of lipid metabolism, appetite (e.g. anorexia nervosa
and bulimia).
referred compounds of the present invention are also useful in treating bleeding
disorde such as hereditary hemorrhagic telangiectasia, dysfunctional uterine bleeding,
and combating hemorrhagic shock.
Preferred compounds of the present invention are useful in disease states where
amenorrhea is advantageous, such as leukemia, endometrial ablations, chronic renal or
hepatic disease or coagulation diseases or disorde.
The compounds of the present invention can be administered to a subject for a
variety of purposes. Except when noted, the terms "subject" or "patient" are used
interchangeably and refe to all mammalian species. For example, the term includes
mammals such as human patients and non-human primates, as well as experimental
animals such as rabbits, rats, and mice, and other animals. Accordingly, the term
"subject" or "patient" as used herein means any mammalian patient or subject to which
the compounds of the invention can be administered. Patients for treatment according to
the methods of the invention preferably are identified using accepted screening methods
to determine risk facto associated with a targeted or suspected disease or condition or to
determine the status of an existing disease or condition in a subject. These screening
methods include, for example, conventional work-ups to determine risk facto that may
be associated with the targeted or suspected disease or condition. These and other routine
methods allow the clinician to select patients in need of therapy using the methods and
formulations of the present invention. A subject "in need thereof is a subject suffering
or suspected to be suffering from a certain condition or disease state treatable by the
methods of the present invention.
As used herein, the term "treating" or "treatment" refe to any indicia of success
in amelioration of an injury, pathology, or condition, including any objective or
subjective parameter such as abatement; inhibition; remission; diminishing of symptoms
making the injury, pathology, or condition more tolerable to the patient; slowing in the
rate of degeneration or decline; making the final point of degeneration less debilitating;
or improving a subject's physical or mental well-being. The treatment or amelioration of
symptoms can be based on objective or subjective paramete; including the results of a
physical examination, neurological examination, and/or psychiatric evaluation.
"Treating" or "treatment of a disease or condition characterized by chronic
inflammation" includes preventing the onset of symptoms in a subject that may be
predisposed to an inflammatory disease but does not yet experience or exhibit symptoms
of the disease (prophylactic treatment), inhibiting the symptoms of the disease (slowing
or arresting its development), providing relief from the symptoms or side-effects of the
disease (including palliative treatment), and/or relieving the symptoms of the disease
(causing regression). Treating or treatment of any disease or condition disclosed herein
includes preventing the onset of symptoms in a subject that may be predisposed to said
disease or condition but does not yet experience or exhibit symptoms of the disease or
condition (prophylactic treatment), inhibiting the symptoms of the disease or condition
(slowing or arresting its development), providing relief from the symptoms or side-effects
of the disease or condition (including palliative treatment), and/or relieving the symptoms
of the disease or condition (causing regression). Accordingly, the term "treating"
includes the administration of the compounds or agents of the present invention to a
subject to prevent or delay, to alleviate, or to arrest or inhibit development of the
symptoms or conditions associated with a disease state. A skilled medical practitioner
will know how to use standard methods to determine whether a patient is suffering from a
disease characterized by chronic inflammation and/or increased cytokine expression.
"Concomitant administration" of a known drug or treatment with a
pharmaceutical composition of the present invention means administration of the drug (or
treatment) and the compound of the present invention at such time that both the known
drug (or treatment) and the composition of the present invention will have a therapeutic
effect. Such concomitant administration can involve concurrent (i.e. at the same time),
prior, or subsequent administration of the drug (or treatment) with respect to the
administration of a compound of the present invention. A peon of ordinary skill in the
art, would have no difficulty determining the appropriate timing, sequence and dosages of
administration for particular drugs and compositions of the present invention. For
example, the compounds of this invention can be used in combination (administered
together or sequentially) with known drugs useful in the treatment of chronic
inflammation.
The present invention provides compositions comprising a compound of the
present invention in combination with a pharmaceutically acceptable carrier. In a
preferred embodiment, the compounds are formulated as Pharmaceuticals to diseases
associated with chronic inflammation
In general, compounds of the present invention can be administered as
pharmaceutical compositions by any method known in the art for administering
therapeutic drugs including oral, buccal, topical, systemic (e.g., transdermal, intranasal,
or by suppository), or parenteral (e.g., intramuscular, subcutaneous, or intravenous
injection). Compositions can take the form of tablets, pills, capsules, semisolids,
powde, sustained release formulations, solutions, suspensions, emulsions, syrups,
elixi, aerosols, or any other appropriate compositions; and comprise at least one
compound of this invention in combination with at least one pharmaceutically acceptable
excipient. Suitable excipients are well known to peons of ordinary skill in the art, and
they, and the methods of formulating the compositions, can be found in such standard
references as Alfonso AR: Remington's Pharmaceutical Sciences, 17th ed., Mack
Publishing Company, Easton PA, 1985. Suitable liquid carrie, especially for injectable
solutions, include water, aqueous saline solution, aqueous dextrose solution, and glycols.
In some embodiments of the present invention, compounds of the present invention
suitable for use in the practice of this invention can be administered either singly or in
combination with at least one other compound of this invention. Compounds suitable for
use in the practice of the present invention can also be administered with at least one
other conventional therapeutic agent for the disease being treated.
Aqueous suspensions of the invention can contain a compound of the present
invention in admixture with excipients suitable for the manufacture of aqueous
suspensions. Such excipients can include, for example, a suspending agent, such as
sodium carboxymemylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium
alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispeing or wetting
agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product
of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation
product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethylene
oxycetanol), a condensation product of ethylene oxide with a partial ester derived from a
fatty acid and a hexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensation
product of ethylene oxide with a partial ester derived from fatty acid and a hexitol
anhydride (e.g., polyoxyethylene sorbitan mono-oleate). The aqueous suspension can
also, for example, contain one or more preservatives such as ethyl or n-propyl phydroxybenzoate,
one or more coloring agents, one or more flavoring agents, and one or
more sweetening agents, such as sucrose, aspartame or saccharin. Formulations can be
adjusted for osmolarity.
Oil suspensions can be formulated by suspending a compound of the present
invention in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in
a mineral oil such as liquid paraffin; or a mixture of these. The oil suspensions can
contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol. Sweetening
agents can be added to provide a palatable oral preparation, such as glycerol, sorbitol or
sucrose. These formulations can be preserved by the addition of an antioxidant such as
ascorbic acid. As an example of an injectable oil vehicle, see Minto, J. Pharmacol. Exp.
Ther. 281:93-102,1997. The pharmaceutical formulations of the invention can also be in
the form of oil-in-water emulsions. The oily phase can be a vegetable oil or a mineral oil,
described above, or a mixture of these. Suitable emulsifying agents include naturallyoccurring
gums, such as gum acacia and gum tragacanth, naturally occurring
phosphatides, such as soybean lecithin, este or partial este derived from fatty acids
and hexitol anhydrides, such as sorbitan mono-oleate, and condensation products of these
partial este with ethylene oxide, such as polyoxyethylene sorbitan mono-oleate. The
emulsion can also contain sweetening agents and flavoring agents, as in the formulation
of syrups and elixi. Such formulations can also contain a demulcent, a preservative, or
a coloring agent.
The compound of choice, alone or in combination with other suitable
components, can be made into aerosol formulations (i.e., they can be "nebulized") to be
administered via inhalation. Aerosol formulations can be placed into pressurized
acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.
Formulations suitable for parenteral administration, such as, for example, by
intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, and
subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection
solutions, which can contain antioxidants, buffe, bacterio stats, and solutes that render
the formulation isotonic with the blood of the intended recipient, and aqueous and nonaqueous
sterile suspensions that can include suspending agents, solubilize, thickening
agents, stabilize, and preservatives. Among the acceptable vehicles and solvents that
can be employed are water and Ringer's solution, an isotonic sodium chloride. In
addition, sterile fixed oils can conventionally be employed as a solvent or suspending
medium. For this purpose any bland fixed oil can be employed including synthetic
mono- or diglycerides. In addition, fatty acids such as oleic acid can likewise be used in
the preparation of injectables. These solutions are sterile and generally free of
undesirable matter. Where the compounds are sufficiently soluble they can be dissolved
directly in normal saline with or without the use of suitable organic solvents, such as
propylene glycol or polyethylene glycol. Dispeions of the finely divided compounds
can be made-up in aqueous starch or sodium carboxymethyl cellulose solution, or in
suitable oil, such as arachis oil. These formulations can be sterilized by conventional,
well known sterilization techniques. The formulations can contain pharmaceutically
acceptable auxiliary substances as required to approximate physiological conditions such
as pH adjusting and buffering agents, toxicity adjusting agents, e.g., sodium acetate,
sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The
concentration of compounds of the present invention in these formulations can vary
widely, and will be selected primarily based on fluid volumes, viscosities, body weight,
and the like, in accordance with the particular mode of administration selected and the
patient's needs. For IV administration, the formulation can be a sterile injectable
preparation, such as a sterile injectable aqueous or oleaginous suspension. This
suspension can be formulated according to the known art using those suitable dispeing
or wetting agents and suspending agents. The sterile injectable preparation can also be a
sterile injectable solution or suspension in a nontoxic parenterally-acceptable diluent or
solvent, such as a solution of 1,3-butanediol. The formulations of commends can be
presented in unit-dose or multi-dose sealed containe, such as ampules and vials.
Injection solutions and suspensions can be prepared from sterile powde,
granules, and tablets of the kind previously described.
Compounds suitable for use in the practice of this invention can be administered
orally. The amount of a compound of the present invention in the composition can vary
widely depending on the type of composition, size of a unit dosage, kind of excipients,
and other facto well known to those of ordinary skill in the art.
Pharmaceutical formulations for oral administration can be formulated using
pharmaceutically acceptable carrie well known in the art in dosages suitable for oral
administration. Such carrie enable the pharmaceutical formulations to be formulated in
unit dosage forms as tablets, pills, powder, dragees, capsules, liquids, lozenges, gels,
syrups, slurries, suspensions, etc. suitable for ingestion by the patient. Formulations
suitable for oral administration can consist of (a) liquid solutions, such as an effective
amount of the packaged nucleic acid suspended in diluents, such as water, saline or PEG
400; (b) capsules, sachets or tablets, each containing a predetermined amount of the
active ingredient, as liquids, solids, granules or gelatin; (c) suspensions in an appropriate
liquid; and (d) suitable emulsions.
Pharmaceutical preparations for oral use can be obtained through combination of
the compounds of the present invention with a solid excipient, optionally grinding a
resulting mixture, and processing the mixture of granules, after adding suitable additional
compounds, if desired, to obtain tablets or dragee cores. Suitable solid excipients are
carbohydrate or protein fille and include, but are not limited to suga, including
lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other
plants; cellulose such as methyl cellulose, hydroxymethyl cellulose,
hydroxypropylmethyl-cellulose or sodium carboxymethylcellulose; and gums including
arabic and tragacanth; as well as proteins such as gelatin and collagen. If desired,
disintegrating or solubilizing agents can be added, such as the cross-linked polyvinyl
pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate. Tablet forms
can include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn
starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc,
magnesium stearate, stearic acid, and other excipients, colorants, fille, binde, diluents,
buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating
agents, and pharmaceutically compatible carrie. Lozenge forms can comprise the
active ingredient in a flavor, e.g., sucrose, as well as pastilles comprising the active
ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions,
gels, and the like containing, in addition to the active ingredient, carrie known in the
art.
The compounds of the present invention can also be administered in the form of
suppositories for rectal administration of the drug. These formulations can be prepared
by mixing the drug with a suitable non-irritating excipient which is solid at ordinary
temperatures but liquid at the rectal temperatures and will therefore melt in the rectum to
release the drug. Such materials are cocoa butter and polyethylene glycols.
The compounds of the present invention can also be administered by intranasal,
intraocular, intravaginal, and intrarectal routes including suppositories, insufflation,
powde and aerosol formulations (for examples of steroid inhalants, see Rohatagi, J.
Clin. Pharmacol. 35:1187-1193,1995; T)Vfa,Ann. Allergy Asthma Immunol. 75:107-111,
1995).
The compounds of the present invention can be administered in sustained or
controlled release dosage forms (e.g., employing a slow release bioerodable delivery
system), including depot injections, osmotic pumps (such as the Alzet implant made by
Alza), pills, transdermal and transcutaneous (including electrotransport) patches, and the
like, for prolonged administration at a predetermined rate, preferably in unit dosage forms
suitable for single administration of precise dosages. The compositions will typically
include a conventional pharmaceutical carrier or excipient and a compound of the
invention. In addition, these compositions can include other active agents, carrie,
adjuvants, and the like.
The compounds of the present invention can be delivered transdermally, by a
topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels,
creams, ointments, pastes, jellies, paints, powde, and aerosols.
Encapsulating materials can also be employed with the compounds of the present
combination with an encapsulating material as a formulation, with or without other
carrie. For example, the compounds of the present invention can also be delivered as
microspheres for slow release in the body.
In another embodiment, the compounds of the present invention can be delivered
by the use of liposomes which fuse with the cellular membrane or are endocytosed. By
using liposomes, particularly where the liposome surface carries ligands specific for
target cells, or are otherwise preferentially directed to a specific organ, one can focus the
delivery of the compound into the target cells in vivo. (See, e.g., Al-Muhammed, J.
Microencapsul. 13:293-306, 1996; Chonn, Curr. Opin. Biotechnol. 6:698-708, 1995;
Ostro, Am. J. Hosp. Pharm. 46:1576-1587,1989).
In other cases, the preferred preparation can be a lyophilized powder which can
contain, for example, any or all of the following: 1 mM-50 mM histidine, 0.1%-2%
sucrose, 2%-7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior
to use.
A pharmaceutical composition of the invention can optionally contain, in addition
to a compound of the present invention, at least one other therapeutic agent useful in the
treatment of a disease or condition associated with chronic inflammation.
The pharmaceutical compositions are generally formulated as sterile, substantially
isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations
of the U.S. Food and Drug Administration.
The present invention provides, inter alia, methods of inhibiting cytokine
expression, e.g., IL-6 expression, in a subject for the treatment of diseases and conditions
associated with increased cytokine activity. In an exemplary embodiment of the present
invention, a skilled practitioner will treat a subject having a disease associated with
chronic inflammation with the compounds of the present invention.
For treatment purposes, the compositions or compounds disclosed herein can be
administered to the subject in a single bolus delivery, via continuous delivery (e.g.,
continuous transdermal, mucosal, or intravenous delivery) over an extended time period,
or in a repeated administration protocol (e.g., by an hourly, daily or weekly, repeated
administration protocol). The pharmaceutical formulations of the present invention can
be administered, for example, one or more times daily, 3 times per week, or weekly. In
an exemplary embodiment of the present invention, the pharmaceutical formulations of
the present invention are orally administered once or twice daily.
In this context, a therapeutically effective dosage of the biologically active
agent(s) can include repeated doses within a prolonged treatment regimen that will yield
clinically significant results to alleviate one or more symptoms or detectable conditions
associated with increased cytokine activity. Determination of effective dosages in this
context is typically based on animal model studies followed up by human clinical trials
and is guided by determining effective dosages and administration protocols that
significantly reduce the occurrence or severity of targeted exposure symptoms or
conditions in the subject. Suitable models in this regard include, for example, murine,
rat, and other accepted animal model subjects known in the art. Alternatively, effective
dosages can be determined using in vitro models. Using such models, only ordinary
calculations and adjustments are typically required to determine an appropriate
concentration and dose to administer a therapeutically effective amount of the
biologically active agent(s) (e.g., amounts that are intranasally effective, transdermally
effective, intravenously effective, or intramuscularly effective to elicit a desired
response). In alternative embodiments, an "effective amount" or "therapeutically
effective dose" or "pharmaceutically effective dose" of the biologically active agent(s)
will simply inhibit or enhance one or more selected biological activity(ies) correlated
with a disease or condition, as set forth above, for either therapeutic or diagnostic
purposes.
The actual dosage of biologically active agents will of coue vary according to
facto such as the extent of exposure and particular status of the subject (e.g., the
subject's age, size, fitness, extent of symptoms, susceptibility facto, etc), time and route
of administration, as well as other drugs or treatments being administered concurrently.
Dosage regimens can be adjusted to provide an optimum prophylactic or therapeutic
response. By "therapeutically effective dose" or "pharmaceutically effective dose" is
meant a dose that produces effects for which it is administered. More specifically, a
therapeutically or pharmaceutically effective dose of the compound(s) of the invention
preferably alleviates symptoms, complications, or biochemical indicia of diseases
associated with increased cytokine activity. The exact dose will depend on the purpose
of the treatment, and will be ascertainable by one skilled in the art using known
techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (Vols. 1-3, 1992); Lloyd,
1999, The Art, Science, and Technology of Pharmaceutical Compounding; and Pickar,
1999, Dosage Calculations). A therapeutically effective dose or pharmaceutically
effective dose is also one in which any toxic or detrimental side effects of the active agent
is outweighed in clinical terms by therapeutically beneficial effects. It is to be further
noted that for each particular subject, specific dosage regimens should be evaluated and
adjusted over time according to the individual need and professional judgment of the
peon administering or supervising the administration of the compounds.
In an exemplary embodiment of the present invention, unit dosage forms of the
compounds are prepared for standard administration regimens. In this way, the
composition can be subdivided readily into smaller doses at the physicians direction. For
example, unit dosages can be made up in packeted powde, vials or ampoules and
preferably in capsule or tablet form.
It is undetood that the effective dosage of the active compounds of this
invention can 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 facto related to the individual being treated. For treating
atherosclerosis, myocardial infarction, congestive heart failure, arthritis and/or
inflammatory bowel disease, generally satisfactory results may be obtained, for example,
when the compounds of this invention are administered to the individual in need at a
daily dosage of from about 0.1 mg to about 1 mg per kilogram of body weight, preferably
administered in divided doses two to six times per day, or in a sustained release form.
For most large mammals, the total daily dosage is from about 3.5 mg to about 140 mg,
preferably from about 3.5 to about 5 mg. In the case of a 70 kg human adult, the total
daily dose will generally be from about 7 mg to about 70 mg and may be adjusted to
provide the optimal therapeutic result.
After a pharmaceutical comprising a compound of the present invention has been
formulated in a suitable carrier, it can be placed in an appropriate container and labeled
for treatment of, for example, a disorder associated with chronic inflammation.
Additionally, another pharmaceutical comprising at least one other therapeutic agent
aseful in the treatment of disorder associated with chronic inflammation can be placed in
the container as well and labeled for treatment of the indicated disease. Alternatively, a
single pharmaceutical comprising a compound of the present invention and at least one
other therapeutic agent useful in the treatment of a disorder associated with chronic
inflammation can be placed in an appropriate container and labeled for treatment. For
administration of pharmaceuticals comprising compounds of the present invention and of
Pharmaceuticals comprising, in a single pharmaceutical, compounds of the present
invention and at least one other therapeutic agent useful in the treatment of disorder
associated with chronic inflammation , such labeling would include, for example,
instructions concerning the amount, frequency and method of administration. Similarly,
for administration of multiple pharmaceuticals provided in the container, such labeling
would include, for example, instructions concerning the amount, frequency and method
of administration of each pharmaceutical.
EXAMPLES
Example 1: Synthesis of 4-{[(6)-8-fluoro-6-methylphenanthridin-5(6//)-
y 1] carbony 1} pheno 1
Step A: 8-fluoro-5-(4-methoxvbenzovlV6-methvl-5.6-dihydrophenanthridine - A
solution of 8-fluoro-6-methyl-5,6-dihydrophenanthridine (O.OSg, 0.23 mmol), 4-
methoxybenzoyl chloride (0.04 g, 0.23 mmol) and 0.08 mL of diisopropylethylamine in 5
mL of THF was stirred at ambient temperature overnight. The reaction mixture was
partitioned with IN NaOH. The organic phase was washed with brine and dried
(NaaSO. The reaction mixture was purified by flash chromatography (silica gel 60,
methylene chloride) to give the product as a white solid (O.OSg, 40% yield). MS (ESI)
Step B: 4-([(6/?V8-fluoro-6-methvlphenanthridin-5(6//)-vllcarbonvUphenol - To
a solution cooled to -78°C of 8-fluoro-5-(4-methoxybenzoyl)-6-methyl-5,6-
dihydrophenanthridine (2.3 g, 6.6 mmol) in 50 mL of methylene chloride containing 2.0
mL of cyclohexene under Na atmosphere was added boron tribromide (2.9 mL, 26
mmol). The reaction was allowed to warm to ambient temperature and stir overnight.
The excess boron tribromide was decomposed by the dropwise addition of methanol to
the ice cooled reaction mixture. The reaction mixture was diluted with methylene
chloride and partitioned with 2N HC1. The organic phase was separated and concentrated
in vacuo. The residual product was purified by flash chromatography ( silica gel 60,
methylene chloride/ethyl acetate, 19:1) to give 1.85 g of the racemate. Isomer separated
by chiral prep HPLC (Chiralcel AD, 25X5 cm, 100% EtOH, 12 mL/min) to give 0.536 g
of the product as °C, 99.86% ee);a white solid (m.p. 165 [a]D
.'H NMR (400 MHz, DMSO-d6) 5 ppm 0.86 (s, 1 H) 1.19 (d, .7=6.98 Hz, 3 H) 5.74 (q,
.7=6.73 Hz, 1 H) 6.65 (d, .7=8.79 Hz, 2 H) 6.68 (d, .7=8.28 Hz, 1 H) 7.05 (dt, .7=7.70, 1.42
Hz, 1 H) 7.12 (d, .7=8.54 Hz, 2 H) 7.18 (m, 1 H) 7.27 (td, .7=8.79, 2.85 Hz, 1 H) 7.38 (dd,
.7=9.31, 2.85 Hz, 1 H) 7.91 (dd, .7=7.76, 1.55 Hz, 1 H) 8.04 (dd, J=8.79, 5.43 Hz, 1 H)
9.96 (s, 1 H); .MS (ESI) m/z 334 ([M+H]+);MS (ESI) m/z 332 ([M-H]');
Example 2: Synthesis of 3-{[(6S)-8-fluoro-6-methylphenanthridin-5(6/f)-
yl]carbonyl}phenol
Step A: 8-fluoro-5-(3-methoxybenzoyl)-6-methyl-5.6-dihydrophenanthridine - A
solution of 8-fluoro-6-methyl-5,6-dihydrophenanthridine (O.OSg, 0.23 mmol), 3-
methoxybenzoyl chloride (0.033 mL, 0.23 mmol) and 0.08 mL of diiisopropylethylamine
in 5 mL of THF was stirred at ambient temperature overnight. The reaction mixture was
partitoned with IN NaOH. The organic phase was washed with brine and dried
(Na2SC>4). The reaction mixture was purified by flash chromatography (silica gel 60,
methylene chloride) to give the product as a white solid (0.045g, 56% yield). MS (ESI)
Step B: 3-ir(6y)-8-fluoro-6-methvlphenanthridin-5r6/n-vncarbonvl)phenol - To
a solution cooled to -78°C of 8-fluoro-5-(3-methoxybenzoyl)-6-methyl-5,6-
dihydrophenanthridine (0.39 g, 1.1 mmol) in 5 mL of methylene chloride containing 0.5
mL of cyclohexene under N2 atmosphere was added boron tribromide (0.495 mL, 4.48
mmol). The reaction was allowed to warm to ambient temperature and stir overnight.
The excess boron tribromide was decomposed by the dropwise addition of methanol to
the ice cooled reaction mixture. The reaction mixture was diluted with methylene
41
chloride and partitioned with 2N HC1. The organic phase was separated and concentrated
in vacuo. The residual product was purified by flash chromatography ( silica gel 60,
methylene chloride/ethyl acetate, 19:1) to give the product (0.318 g, 85% yield) as a
white solid. The pure enantiomer was isolated by chiral HPLC (Xterra MS CIS,
ethanol/hexane 1:1) to give the product as a white solid (0.047 g, 97.6% ee).
mp sinte 92, melts 138 °C; [phenol - To a solution cooled to -78°C of 8-fluoro-5-(3-fluoro-4-
methoxybenzoyl)-6-methyl-5,6-dihydrophenanthridine (0.191 g, 0.52 mmol) in 5 mL of
methylene chloride containing 0.2 mL of cyclohexene under N2 atmosphere was added
45
boron tribromide (0.231 mL, 2.09 mmol). The reaction was allowed to warm to ambient
temperature and stir overnight. The excess boron tribromide was decomposed by the
dropwise addition of methanol to the ice cooled reaction mixture. The reaction mixture
was diluted with methylene chloride and partitioned with 2N HC1. The organic phase
was separated and concentrated in vacuo. The residual product was purified by flash
chromatography ( silica gel 60, methylene chloride/ethyl acetate, 19:1) to give the
product mixture (0.174 g). The chirally pure enantiomer was isolated by chiral HPLC
(Chiralpak AD, 4.6X250 mm, ethanol/hexane 1:1) to give the product as a white solid
25 = -676° (c = 0.010G/ML, MeOH); !H NMR (400 MHz, DMSO-d6) 6 ppm 1.19 (d,
.7=6.92 Hz, 3 H) 5.74 (q, J=6.83 Hz, 1 H) 6.73 (d, .7=7.69 Hz, 1 H) 6.84 (m, 2 H) 7.08 (m,
2 H) 7.22 (dt, .7=7.62, 1.15 Hz, 1 H) 7.27 (dt, 1 H) 7.39 (dd, ,7=9.22, 2.56 Hz, 1 H) 7.93
(dd, .7=7.81, 1.41 Hz, 1 H) 8.05 (dd, .7=8.71, 5.38 Hz, 1 H); MS (ESI) m/z 352 ([M+H]+);
MS (ESI) m/z 350 ([M-H]').
Example 10: Synthesis of 2-fluoro-4-{[(65)-8-fluoro-6-methylphenanthridin-
5(6//>yl]carbonyl}phenol.
Isolated in the above chiral prep (0.027 g, 99.8% ee). !H NMR (400 MHz,
DMSO-d6) 5 ppm 0.84 (s, 1 H) 1.19 (d, .7=6.92 Hz, 3 H) 5.73 (q, .7=6.66 Hz, 1 H) 6.76
(m, 2 H) 6.85 (m, 1 H) 7.07 (m, 2 H) 7.21 (dt, .7=7.56, 1.02 Hz, 1 H) 7.27 (dt, .7=8.84,
2.82 Hz, 1 H) 7.38 (dd, .7=9.22, 2.82 Hz, 1 H) 7.93 (dd, .7=7.81, 1.15 Hz, 1 H) 8.05 (dd,
.7=8.71, 5.64 Hz, 1 H); MS (ESI) m/z 352 ([M+H]+); MS (ESI) m/z 350 ([M-H]').
Example 11: Synthesis of 2-chloro-5-{[(6/?)-8-fluoro-6-methylphenanthridin-
5 (6//)-yl]carbonyl} phenol
Step A: 5-(4-chloro-3-methoxvbenzovlV8-fluoro-6-methvl-5.6-
dihvdrophenanthridine - A solution of 8-fluoro-6-methylphenanthridine (0.211 g, 1.0
mmol) and 4-chloro-3-methoxybenzoyl chloride (0.205 g, 1.0 mmol) in 5 mL of
methylene chloride was slowly added over 1 hour to a stirred solution of (S)-2-methyl-
CBS-oxazaborolidine (0.2 mL, 1M solution in toluene) and borane-methyl sulfide
complex (0.7 mL,lM solution in methylene chloride). The reaction mixture was stirred
46
at ambient temperature overnight. The reaction mixture was partitioned with IN NaOH.
The organic phase was washed with brine and dried (NaiSO. The crude product was
purified by flash chromatography (silica gel 60, methylene chloride) to give the product
as a white solid (0.087 g, 29% of theory); MS (ESI) m/z 382/384 ([M+H]+).
Step B: 2-chloro-5-ir(6/?V8-fluoro-6-rnethvlphenanthridin-5(6//)-
vl]carbonvl}phenol - To a solution cooled to -78°C of 8-fluoro-5-(4-chloro-3-
methoxybenzoyl)-6-methyl-5,6-dihydrophenanthridine (0.076 g, 0.2 mmol) in 5 mL of
methylene chloride containing 0.5 mL of cyclohexene under N2 atmosphere was added
boron tribromide (0.088 mL, 0.8 mmol). The reaction was allowed to warm to ambient
temperature and stir overnight. The excess boron tribromide was decomposed by the
dropwise addition of methanol to the ice cooled reaction mixture. The reaction mixture
was diluted with methylene chloride and partitioned with 2N HC1. The organic phase
was separated and concentrated in vacuo. The residual product was purified by flash
chromatography ( silica gel 60, methylene chloride/ethyl acetate, 19:1) to give the
product mixture (0.70 g). The pure enantiomer was isolated by chiral HPLC (Chiralpak
AD, 4.6X250 mm, acetonitrile/ ethanol, 9:1) to give the product as a white solid (0.015 g,
99.8% ee).
'H-NMR (400 MHz, DMSO-d6) 8 ppm 1.19 (d, 3 H) 5.74 (broad s, 1 H) 6.65 (broad d, 1
H) 6.77 (broad s, 1 H) 6.91 (s, 1H) 7.23 (t, 1 H) 7.5 (m, 3 H)) 7.39 (dd, 1 H) 7.93 (dd, 1
H) 8.05 (dd,l H) 10.42 (s, 1H); MS (ESI) m/z 368/370 ([M+H]+); MS (ESI) m/z 366/368
([M-H]').
Example 12: Synthesis of 2-chloro-5-{[(6S)-8-fluoro-6-methylphenanthridin-
5(6/f)-yl]carbonyl}phenol.
Isolated from the above chiral prep to give 0.02 g (99.8%ee) of the pure
enantiomer. [a]D
25 = +577° (c = 0.0082G/ML, MeOH); MS (ESI) m/z 368/370 ([M+H]+);
MS (ESI) m/z 366/368 ([M-H]').
Example 13: Synthesis of 2-bromo-4-{[(6/?)-8-fluoro-6-methylphenanthridin-
5(6//)-yl]carbonyl}phenol.
Step A: 5-(3-bromo-4-methoxvbenzovl)-8-fluoro-6-methyl-5,6-
dihvdrophenanthridine - A solution of 8-fluoro-6-methylphenanthridine (0.211 g, 1.0
mmol) and 3-bromo-4-methoxybenzoyl chloride (0.249 g, 1.0 mmol) in 5 mL of
methylene chloride was slowly added over 1 hour to a stirred solution of (S)-2-methyl-
CBS-oxazaborolidine (0.2 mL, 1M solution in toluene) and borane-methyl sulfide
complex (0.7 mL,lM solution in methylene chloride). The reaction mixture was stirred
at ambient temperature overnight. The reaction mixture was partitioned with IN NaOH.
The organic phase was washed with brine and dried (NaaSC). The crude product was
purified by flash chromatography (silica gel 60, methylene chloride) to give the product
as a white solid (0.180 g, 42% of theory); MS (ESI) m/z 426/428 ([M+H]+).
Step B: 2-bromo-4-([(6V8-fluoro-6-methvlphenanthridin-5(6/f)-
yl]carbonvl}phenol - To a solution cooled to -78°C of 8-fluoro-5-(3-bromo-4-
methoxybenzoyl)-6-methyl-5,6-dihydrophenanthridine (0.175 g, 0.41 mmol) in 5 mL of
methylene chloride containing 0.1 mL of cyclohexene under Na atmosphere was added
boron tribromide (0.181 mL, 1.63 mmol). The reaction was allowed to warm to ambient
temperature and stir overnight. The excess boron tribromide was decomposed by the
dropwise addition of methanol to the ice cooled reaction mixture. The reaction mixture
was diluted with methylene chloride and partitioned with 2N HC1. The organic phase
was separated and concentrated in vacuo. The residual product was purified by flash
chromatography ( silica gel 60, methylene chloride/ethyl acetate, 19:1) to give the
product mixture (0.160 g). The chirally pure enantiomer was isolated by chiral HPLC
(Chiralpak AD, 4.6X250 mm, acetonitrile/ethanol, 9:1) to give the product as a white
solid (0.041 g, 99.98% ee). >H NMR (400 MHz, DMSO-d6) 8 ppm 1.19 (d, 3 H) 5.74 (m,
1 H) 6.74 (d, 1 H) 6.77 (d, 1 H) 7.01 (dd, 1H) 7.21 dt, 1 H) 7.25 (m, 3 H) 7.39 (dd, 1H)
7.42 (d, 1H) 7.93 (dd, 1 H) 8.05 (dd,l H) 10.90 (s, 1H);MS (ESI) m/z 412/414 ([M+H]+);
MS (ESI) m/z 410/412 ([M-H]").
Example 14: Synthesis of 2-bromo-4-{[(65)-8-fluoro-6-methylphenanthridin-
5 (6/f)-yl] carbonyl} phenol.
Isolated from the above chiral prep to give 0.049 g (99.8%ee) of the title compound.
25 = +533° (c = 0.0104G/ML, MeOH); 1H NMR (400 MHz, DMSO-d6) 8 ppm 1.19
(d, 3 H) 5.74 (m, 1 H) 6.74 (d, 1 H) 6.77 (d, 1 H) 7.01 (dd, 1H) 7.22 dt, 1 H) 7.25 (m, 3
H) 7.39 (dd, 1H) 7.43 (d, 1H) 7.93 (dd, 1 H) 8.05 (dd,l H).10.89 (s, 1H); MS (ESI) m/z
412/414 ([M+H]+); MS (ESI) m/z 410/412 ([M-H]-).Example 15: Synthesis of 4-bromo-
3-{[(6J?)-8-fluoro-6-methylphenanthridin-5(6//)-yl]carbonyl}phenol.
Step A: 5-(2-bromo-5-methoxvbenzovl)-8-fluoro-6-methyl-5,6-
dihydrophenanthridine - A solution of 8-fluoro-6-methylphenanthridine (0.211 g, 1.0
mmol) and 2-bromo-5-methoxybenzoyl chloride (0.231 g, 1.0 mmol) in 5 mL of
methylene chloride was slowly added over 1 hour to a stirred solution of (S)-2-methyl-
CBS-oxazaborolidine (0.2 mL, 1M solution in toluene) and borane-methyl sulfide
complex (0.7 mL,lM solution in methylene chloride). The reaction mixture was stirred
at ambient temperature overnight. The reaction mixture was partitioned with IN NaOH.
The organic phase was washed with brine and dried (Na2SO4). The crude product was
purified by flash chromatography (silica gel 60, methylene chloride) to give the product
as a white solid (0.258 g, 60% of theory); MS (ESI) m/z 426/428 ([M+H]+).
Step B: 4-bromo-3-([(6/?)-8-fluoro-6-methvlphenanthridin-5(6fflylJcarbonyUphenol
To a solution cooled to -78°C of 8-fluoro-5-(2-bromo-5-
methoxybenzoyl)-6-methyl-5,6-dihydrophenanthridine (0.243 g, 0.57 mmol) in 5 mL of
methylene chloride containing 0.2 mL of cyclohexene under N2 atmosphere was added
boron tribromide (0.252 mL, 2.28 mmol). The reaction was allowed to warm to ambient
temperature and stir overnight. The excess boron tribromide was decomposed by the
dropwise addition of methanol to the ice cooled reaction mixture. The reaction mixture
was diluted with methylene chloride and partitioned with 2N HC1. The organic phase
was separated and concentrated in vacuo. The residual product was purified by flash
chromatography ( silica gel 60, methylene chloride/ethyl acetate, 19:1) to give the
product mixture (0.210 g). The pure enantiomer was isolated by chiral HPLC (Whelk-01
(S,S), ethanol/hexane 1:1) to give the product as a white solid (0.080 g, 99.8% ee).lH
NMR (400 MHz, DMSO-d6) 8 ppm 1.19 (d, .7=6.98 Hz, 3 H) 6.06 (d, .7=5.69 Hz, 1 H)
6.70 (m, 2 H) 6.85 (dd, 1 H) 7.03 (m, 1 H) 7.22 (m, 2 H) 7.44 (m, 2 H) 7.88 (d, .7=7.76
Hz, 1 H) 7.99 (m, 2 H) 10.03 (s, 1 H)
MS (ESI) m/z 412/414 ([M+H]+); MS (ESI) m/z 410/412 ([M-H]").
Example 16: Synthesis of 4-bromo-3-{[(6S)-8-fluoro-6-methylphenanthridin-
5(6//)-yl]carbonyl}phenol.
Isolated in the above chiral prep to give 0.055 g (98.6% ee) of the title compound.
25 = +476° (c = 0.0104G/ML, MeOH); MS (ESI) m/z 412/414 ([M+H]+); MS (ESI)
Example 17: Synthesis of 4-{[(6/?)-8-fluoro-6-methylphenanthridin-5(6//)-
yl]carbonyl}-2-methoxyphenol.
A solution of 8-fluoro-6-methylphenanthridine (0.211 g, 1.0 mmol) and 3-
methoxy-4-hydroxybenzoyl chloride (0.168 g, 1.0 mmol) in 5 mL of methylene chloride
was slowly added over 1 hour to a stirred solution of (S)-2-methyl-CBS-oxazaborolidine
(0.2 mL, 1M solution in toluene) and borane-methyl sulfide complex (0.7 mL,lM
solution in methylene chloride). The reaction mixture was stirred at ambient temperature
overnight. The reaction mixture was partitioned with IN NaOH. The organic phase was
washed with brine and dried (NaaSC). The crude product was purified by flash
chromatography (silica gel 60, methylene chloride) to give the product mixture (0.053 g).
The pure enantiomer was isolated by chiral HPLC (AD, acetonitrile/ethanol, 9:1) to give
the product as a white solid (0.033 g, 99.8% ee). 'H NMR (400 MHz, DMSO-d6) 5 ppm
1.20 (d, .7=6.98 Hz, 3 H) 3.61 (s, 3 H) 5.76 (q, .7=6.64 Hz, 1 H) 6.64 (m, 2 H) 6.71 (d,
.7=8.28 Hz, 1 H) 6.89 (d, .7=1.03 Hz, 1 H) 7.06 (td, .7=7.70,1.42 Hz, 1 H) 7.20 (td, .7=7.57,
1.16 Hz, 1 H) 7.26 (td, .7=8.79, 2.85 Hz, 1 H) 7.39 (dd, .7=9.31, 2.85 Hz, 1 H) 7.92 (dd,
.7=8.02,1.29 Hz, 1 H) 8.05 (dd, J=8.67, 5.56 Hz, 1 H); MS (ESI) m/z 364 ([M+H]+);
MS (ESI) m/z 362 ([M-H]').
Example 18: Synthesis of 4-{[(6S)-8-fluoro-6-methylphenanthridin-5(6//)-
yl] carbonyl}-2-methoxy phenol
Isolated from the above chiral prep to give 0.02 g (98.6 % ee) of the title
compound.
25 = +493° (C = 0.008G/ML, MeOH); H NMR (400 MHz, DMSO-d6) 8 ppm 1.19 (d,
J=6.73 Hz, 3 H) 3.61 (s, 3 H) 5.76 (q, J=6.64 Hz, 1 H) 6.64 (m, 2 H) 6.71 (d, J=8.02 Hz,
1 H) 6.88 (d, .7=1.03 Hz, 1 H) 7.06 (td, .7=7.70, 1.42 Hz, 1 H) 7.20 (td, .7=7.57, 1.16 Hz, 1
50
H) 7.26 (td, .7=8.79, 2.85 Hz, 1 H) 7.39 (dd, .7=9.31, 2.85 Hz, 1 H) 7.92 (dd, 7=7.89,
Hz, 1 H) 8.05 (dd, J=8.79, 5.43 Hz, 1 H) 9.63 (s, 1 H); MS (ESI) m/z 364 ([M+H]+); MS
(ESI) m/z 362 ([M-H]').
Example 19: Synthesis of 4-{[(6S)-6-ethyl-8-fluorophenanthridin-5(6//)-
yl] carbonyl } phenol
Step A: 6-Ethvl-8-methvl-phenanthridine - To cold (dry ice /acetone)
ethylmagnesium bromide (20 mL of 1.0 M in THF) was added a mixture of 4, 2'-
difluoro-biphenyl-2-carbonitrile ( 4.6 mmol, 1.0 g) in 10 mL of THF, dropwise (15
min.). Stirring was continued overnight, and the reaction mixture was diluted with 1 0 %
ammonium chloride and ethyl acetate. The organic portion was washed with water and
brine and dried over magnesium sulfate. The solvent was removed and the crude product
flashed (dichloromethane) giving a white solid: mp 74-75 °C (0.44 g, 52%); MS m/z 216
.([M+H]).
Step B: 6-ethvl-8-fluoro-5-(4-methoxvbenzovlV5.6-dihvdrophenanthridine - To a
mixture of borane methyl sulfide complex (2.5 mL of 1 M solution in dichloromethane),
S-2-Methyl-CBS-oxazaborolidine (1 mL of 1 M solution in toluene) and anhydrous
dichloromethane (25 mL) was added a solution of 6-Ethyl-8-methyl-phenanthridine
(0.906 g, 5 mmol) and 4-Methoxybenzoyl chloride (0.938 g, 5.5 mmol) in
dichloromethane (10 mL), dropwise. The reaction mixture was stirred at room
temperature overnight. Sodium hydroxide (10 mL of 2 N solution) was added, the
organic portion dried over anhydrous magnesium sulfate and the solvent removed to
provide a dark liquid. The crude material was purified by flash chromatography
(dichloromethane) to afford a white solid (0.95 g, 52%): mp 71-72 °C; MS m/z 362
Step C: 4-1 f(6S-6-ethvl-8-fluorophenanthridin-5 (6/7)- vljcarbonyl } phenol - To a
mixture of neat boron tribromide (1.2 mL) and cyclohexene (2 mL) in anhydrous
dichloromethane (20 mL) cooled with dry ice/isopropanol was added a mixture of 6-
ethyl-8-fluoro-5-(4-methoxybenzoyl)-5,6-dihydrophenanthridine (0.90 g, 2.5 mmol) in
dichloromethane (10 mL), dropwise (20 min.) The reaction mixture was stirred overnight
as the temperature rose to room temperature. The excess boron tribromide was
decomposed by adding a small quantity of methanol, followed by 2 N HC1 (5 mL). The
organic portion was dried over anhydrous magnesium sulfate, the solvent was removed
and the crude product was purified by chromatography (5% ethyl acetatedichloromethane)
to afford a tan solid (0.583g), The compound was purified by chiral
HPLC to give 0.220 g of title compound, mp 103-104 °C; H NMR (DMSO-d6): 5 0.86
(3 H, t, .7=7.4 Hz), 1.33-1.43 (1H, m), 1.45-1.54 (1 H, m), 5.57-5.61 (1 H, m), 6.62 (2 H,
d, .7=8.8 Hz), 6.68 (1H, d, J=8.0 Hz), 7.04-7.08(3 H, m), 7.17-7.21 (1 H, m), 7.25-7.30 (1
H, m), 7.38 (1 H, dd, .7=9.3 Hz, .7=2.7 Hz), 7.91 (1H, dd, .7=7.8 Hz, ,7=1.2 Hz), 8.04 (1H,
dd, J=8.7 Hz, J=5.5 Hz), 9.92 (1H, br s); MS m/z 348 ([M+H]+).
Example 20: Synthesis of 4-{[(6fl)-6-ethyl-8-fluorophenanthridin-5(6//)-
yl] carbony 1} phenol
The minor isomer was also isolated in the above separation. (0.100 g): mp 105-
106; 'HNMR (DMSO-d6): 6 0.86 (3 H, t, .7=7.4 Hz), 1.33-1.43 (1 H, m), 1.45-1.55 (1 H,
m), 5.57-5.61 (1 H, m), 6.62 (2 H, d, .7=8.8 Hz), 6.68 (1 H, d, .7=7.9 Hz), 7.04-7.09 (3 H,
m), 7.17-7.21 (1 H, m), 7.25-7.30 (1 H, m), 7.38 (1 H, dd, .7=9.2 Hz, .7=2.7 Hz), 7.91 (1
H, dd,, J=7.9 Hz, J=1.3 Hz), 8.04 (1 H, dd, .7=8.7 Hz, J=5.5 Hz), 9.92 (1 H, br s);
MS m/z 348 ([M+H]+).
Example 21: Synthesis of 3-{[(6/?)-6-ethyl-8-fluorophenanthridin-5(6//)-
yl] carbonyl} phenol
To a mixture of borane methyl sulfide complex (0.6 mL of 1 M solution in
dichloromethane), S-2-Methyl-CBS-oxazaborolidine (0.2 mL of 1 M solution in toluene)
and anhydrous dichloromethane (5 mL) was added a solution of 6-Ethyl-8-methylphenanthridine
(0.225 g, 1 mmol) and 3-methoxybenzoyl chloride (0.188 g, 1.1 mmol) in
dichloromethane (5 mL), dropwise. The reaction mixture was stirred at room
temperature overnight. Sodium hydroxide (10 mL of 2 N solution) was added, the
organic portion dried over anhydrous magnesium sulfate and the solvent removed to
provide a dark liquid. The crude material was purified by chromatography
(dichloromethane) to afford a white solid (.08 g, 22%). The material was taken forward
without getting analyses. To a mixture of neat boron tribromide (.2 mL) and cyclohexene
(0.2 mL) in anhydrous dichloromethane (10 mL) cooled with dry ice/isopropanol was
added a mixture the methoxy derivative above 0.080 g, 0.2 mmol) in dichloromethane
(10 mL), dropwise (20 min.) The reaction mixture was stirred overnight as the
temperature rose to room temperature. The excess boron tribromide was decomposed by
adding a small quantity of methanol, follwed by 2 N HC1 (5 mL). The organic portion
was dried over anhydrous magnesium sulfate, the solvent was removed and the crude
product was purified by chromatography (5% ethyl acetate-dichloromethane) to afford a
solid (0.045g), which was purified by chiral HPLC to give the major isomer: 0.030 g: mp
101-102; 'H NMR (DMSO-d6): 8 0.86 (3 H, t, J=7.3 Hz), 1.31-1.42 (1 H, m), 1.46-1.54
(1 H, m), 5.59 (1 H, br s), 6.56 (1 H, d, .7=7.4 Hz), 6.61 (1 H, s), 6.76 (2 H, dd, J=8.1 Hz,
.7=1.7 Hz), 7.04-7.09 (2 H, m), 7.19-7.23 (1 H, m), 7.26-7.31 (1 H, m), 7.36-7.39 (1 H,
m), 7.92 (1 H, dd, .7=7.9 Hz, .7=1.2 Hz), 8.06 (1 H, dd, .7=8.7 Hz, .7=5.5 Hz), 9.56 (1 H, s);
MS m/z 348 ([M+H]+).
Example 22: Synthesis of 4-{[(67?)-6-ethyl-8-fluorophenanthridin-5(6//)-
yl] carbonyl} benzene-1,3 -diol
Step A: 5-(2.4-dimethoxybenzovlV6-ethvl-8-fluoro-5.6-dihvdrophenanthridine -
To a mixture of borane methyl sulfide complex (0.6 mL of 1 M solution in
dichloromethane), S-2-Methyl-CBS-oxazaborolidine (0.2 mL of 1 M solution in toluene)
and anhydrous dichloromethane (5 mL) was added a solution of 6-Ethyl-8-methylphenanthridine
(0.225 g, 1 mmol) and 2,4-dimethoxybenzoyl chloride (0.221 g, 1.1
mmoles) in dichloromethane (5 mL), dropwise. The reaction mixture was stirred at room
temperature overnight. Sodium hydroxide (10 mL of 2 N solution) was added, the
organic portion dried over anhydrous magnesium sulfate and the solvent removed to
provide a dark liquid. The crude material was purified by chromatography
(dichloromethane) to afford a white solid (0.18 g, 32%): mp 125-126 °C;
MS (ESI) m/z 392 ([M+H]+).
Step B: 4-1 [(6/?V6-ethvl-8-fluorophenanthridin-5(6//)-vl]carbonvUbenzene-1.3-
diol - To a mixture of neat boron tribromide (.2 mL) and cyclohexene (0.2 mL) in
anhydrous dichloromethane (10 mL) cooled with dry ice/isopropanol was added a
solution of 5-(2,4-dimethoxybenzoyl)-6-ethyl-8-fluoro-5,6-dihydrophenanthridine (0.080
g, 0.2 mmol) in dichloromethane (10 mL), dropwise (20 min.) The reaction mixture was
stirred overnight as the temperature rose to room temperature. The excess boron
tribromide was decomposed by adding a small quantity of methanol, then 2 N HC1 was
added (5 mL). The organic portion was dried over anhydrous magnesium sulfate, the
solvent was removed and the crude product was purified by chromatography (5% ethyl
acetate-dichloromethane) to afford a solid (O.OSg), which was submitted for
chromatographic separation of the isome. The major isomer: 0.012 g: mp 98-100; H
NMR (DMSO-d6): 5 0.84 (3 H, t, J=7.3 Hz), 1.34-1.41 (1 H, m), 1.49-1.56 (1 H, m), 5.55
(1 H, br s), 6.10 (1 H, d, ,7=1.9 Hz), 6.14 (1 H, dd, .7=8.4 Hz, ,7=2.2 Hz), 6.85 (1 H, d,
.7=8.3 Hz), 7.05 (1 H, t, J=7.4 Hz), 7.15-7.19 (1 H, m), 7.21-7.26 (1 H, m), 7.31-7.33 (2
H, m), 7.85 (1 H, dd, .7=7.8 Hz, ,7=1.3 Hz), 7.96 (1 H, dd, J=8.7 Hz, J=5.5 Hz), 9.53 (1 H,
s),9.61(lH,s);
MS m/z 364 ([M+H]+).
Example 23: Synthesis of 4-[(6-ethyl-8-fluorophenanthridin-5(6//)-yl) carbonyl]-
3-fluorophenol
Step A: 6-ethyl-8-fluoro-5-(2-fluoro-4-methoxybenzoyl')-5.6-
dihvdrophenanthridine - To a mixture of borane methyl sulfide complex (0.5 mL of 1 M
solution in dichloromethane), S-2-Methyl-CBS-oxazaborolidine (0.2 mL of 1 M solution
in toluene) and anhydrous dichloromethane (5 mL) was added a solution of 6-Ethyl-8-
methyl-phenanthridine (0.225 g, 1 mmol) and 2-fluoro-4-methoxybenzoyl chloride (0.20
g, 1.1 mmol) in dichloromethane (5 mL), dropwise. The reaction mixture was stirred at
room temperature overnight. Sodium hydroxide (10 mL of 2 N solution) was added, the
organic portion dried over anhydrous magnesium sulfate and the solvent removed to
provide a dark liquid. The crude material was purified by chromatography
(dichloromethane) to afford a white solid (0.13 g, 33 %):
MS (ESI) m/z 380 ([M+H]+);
Step B 4-[(6-ethvl-8-fluorophenanthridin-5(6//)-vl) carbonvl]-3-fluorophenol -
To a mixture of neat boron tribromide (0.1 mL) and cyclohexene (0.2 mL) in anhydrous
dichloromethane (10 mL) cooled with dry ice/isopropanol was added a solution of 6-
ethyl-8-fluoro-5-(2-fluoro-4-methoxybenzoyl)-5,6-dihydrophenanthridine (0.120 g, 0.3
54
mmol) in dichlorpmethane (10 mL), dropwise (20 min.) The reaction mixture was stirred
overnight as the temperature rose to room temperature. The excess boron tribromide was
decomposed by adding a small quantity of methanol, then 2 N HC1 was added (5 mL).
The organic portion was dried over anhydrous magnesium sulfate, the solvent was
removed and the crude product was purified by chromatography (10% ethyl acetatedichloromethane)
to afford a solid (0.045g): mp 109-111 °C; 'H NMR (DMSO-d6): 5
0.86 (3 H, t, 7=7.2 Hz), 1.27-1.35 (1 H, m), 1.48-1.54 (1 H, m), 5.69 ( 1 H, br s), 6.33 (1
H, br s), 6.60 (1 H, d, .7=6.7 Hz), 7.06 (1 H, br s), 7.19-7.28 (2 H, m), 7.38 (1 H, d, J=8.0
Hz), 7.89 (1 H, d, .7=7.0 Hz), 8.01 (1 H, dd, .7=8.5 Hz, .7=5.3 Hz), 10.32 (1 H, br s); MS
mtz 366 ([M+H]+).
Example 24: Synthesis of 3-[(3-chloro-6-methylphenanthridin-5(6/7)-
yl)carbonyl]phenol
Step A: 1 -(4'-chloro-2'-fluoro-1.1 '-biphenyl-2-vl)ethanone - 2-Acetylphenyl
boronic acid (5g, 30.5 mmol) and 4-chloro-2-fluoroiodobenzene (8.6 g, 33.5 mmol) were
dissolved in toluene/ethanol mixture (6:1, 175 mL). An aqueous solution of potassium
carbonate (2M, 60 mL) and tetrakis(triphenylphosphine) palladium (0) (1.06 g, 0.91
mmol) were added to the solution and the entire mixture was degassed using vacuum and
stirring with intermittent nitrogen purge. The mixture was heated (85 °C) with stirring
for 14 h. The mixture was allowed to cool and then water was added. The organic phase
was separated from the aqueous phase and the aqueous phase was extracted with ethyl
acetate (3 x 100 mL). The organic phases were combined, dried (NaaSO, filtered, and
concentrated to a crude oil. The product was isolated via flash column chromatography
(Biotage® 40 Mi, 5-20%, methyl tert-butylether in hexane) and afforded 4.07 g (54%) of
the desired product as a yellow oil. MS (El) m/z 248.0/250.0 (M+.);
'H NMR (400 MHz, DMSO-d6) 6 ppm 2.45 (s, 3 H) 7.37 (m, 3 H) 7.44 (m, 1 H) 7.57 (td,
Step B: l-(4'-Chloro-2'-fluoro-biphenvl-2-vlVethylamine - To a stirred solution
of l-(4'-Chloro-2'-fluoro-biphenyl-2-yl)-ethanone (3.2 g, 12.9 mmol) in anhydrous
methanol (200 mL) was added solid ammonium acetate (19.8 g, 257 mmol). The
reaction mixture was heated (60 °C, Ih) followed by the addition of a methanolic solution
55
of sodium cyanoborohydride (1.62g, 25.8 mmol). After 16h, the methanol was removed
in vacua and aqueous ammonium hydroxide was added. The aqueous phase was
extracted with diethyl ether (3 x 200 mL) until the amine was no longer present in the
aqueous phase. The organic phase was then washed with 2N aqueous HC1 (3 x 100 mL)
and the aqueous phases combined. The solid formed during the acid wash was
determined to be the dialkyated amine and was segregated from the aqueous phase.
Aqueous sodium hydroxide was then added to the acidic aqueous phase until the solution
was basic (pH 8-9). The basic aqueous phase was extracted with diethyl ether until the
primary amine was no longer detected in the aqueous phase. The combined organic
phases were dried (Na2SO4), filtered, and concentrated to afford a clear oil that was used
without further purification.
benzylic cation as a result of loss of NH2;
Step C: N-\ 1 -(4'-chloro-2'-fluoro-l. 1 '-biphenvl-2-vl)ethvl1-3-methoxvbenzamide
.To a solution of l-(4'-Chloro-2'-fluoro-biphenyl-2-yl)-ethylamine (1 g, 4 mmol) and
triethylamine (614 uL, 4.4 mmol) in acetonitrile was added 3-methoxybenzoyl chloride
(683 mg, 4 mmol). The reaction was mixed overnight (16 h) on an orbital mixer at room
temperature. The acetonitrile was removed in vacuo and the resulting solid dissolved in
dichloromethane and applied directly to a Biotage® (40Mi) column for flash column
chromatography (5-30% methyl tert-butylether in hexane) resulting in the isolation of
1.22 g (79%) of a white solid, mp 176.2-177.9 °C; 'H NMR (400 MHz, DMSO-d6) 8
ppm 1.31 (d, J=5.69 Hz, 3 H) 3.79 (s, 3 H) 4.96 (m, 1 H) 7.07 (dd, J=8.02, 1.81 Hz, 1 H)
7.16 (d, .7=7.24 Hz, 1 H) 7.36 (m, 7 H) 7.60 (m, 2 H) 8.80 (s, 1 H); MS (ESI) m/z
([M+H]+);Anal. calcd for CazHuClFNCh: C:68.84 H:4.99 N:3.65 Found: C:68.88 H:5.25
N:3.64.
Step D: 3-chloro-5-(3-methoxvbenzovn-6-methyl-5.6-dihvdrophenanthridine -
N-[l-(4'-Chloro-2'-fluoro-biphenyl-2-yl)-ethyl]-3-methoxy-benzamide (1.04 g, 2.7 mmol)
was dissolved in anhydrous THF (10 mL) and the vial was capped and purged with an
inert atmosphere. Lithium bis(trimethylsilyl)amide (4.1 mL, 1M in THF) was added to
the solution and the mixture heated (70 °C). The reaction progress was monitored by
LCMS and heating was discontinued upon the expense of the starting material. The THF
was removed and the resulting residue was purified by flash column chromatography
56
(Biotage® 40 Mi, 30-50% methyl tert-butylether in hexane) affording 537 mg (54%) of
the desired product. MS [(ES+), m/z]: 364/366 [M+H]+, 1 Cl pattern.
Step E: 3-[(3-chloro-6-methvlphenanthridin-5(6//)-yncarbonvl]phenol - To a
solution of (3-chloro-6-methyl-6H-phenanthridin-5-yl)-(3-methoxy-phenyl)-methanone
(484 mg, 1.33 mmol) in dichloromethane was added cyclohexene (900 uL, 8.9 mmol).
The vial was capped and purged with an inert atmosphere followed by the addition of a 1
M solution of boron tribromide in dichloromethane (5.3 mL). The solution was mixed at
room temperature using an orbital shaker. After 3 hou the reaction was cooled in an ice
bath (0 °C) and quenched upon the slow addition of methanol. The clear solution was
concentrated and the resulting residue was immediately purified using a Biotage® 40 Mi
column of prepacked silica gel (90g), eluting with a gradient of 5-30% methyl tertbutylether
in hexane at a flow rate of 50 mL/min. The fractions containing the desired
product were combined and concentrated to afford 397 mg (85%) of a white solid. H
NMR (400 MHz, DMSO-d6) 6 ppm 1.17 (d, .7=6.73 Hz, 3 H) 5.60 (d, J=6.21 Hz, 1 H)
6.66 (d, .7=7.50 Hz, 1 H) 6.69 (s, 1 H) 6.80 (ddd, .7=8.21, 2.52, 0.91 Hz, 1 H) 6.88 (s, 1 H)
7.13 (t, ,7=7.89 Hz, 1 H) 7.25 (dd, .7=8.41, 2.20 Hz, 1 H) 7.38 (m, 3 H) 7.94 (d, .7=8.54
Hz, 2 H) 9.63 (s, 1 H); MS (ESI) m/z 350/352 ([M+H]+); MS (ESI) m/z 348/350 ([M-H]').
Example 25: Synthesis of 3-{[(6/?)-3-chloro-6-methylphenanthridin-5(6/7)-
y 1] carbony 1} phenol
The enantiome of 3-[(3-chloro-6-methylphenanthridin-5(6//)-
yl)carbonyl]phenol (367 mg, 1.05 mmol) were separated by automated, preparative,
normal phase, chiral chromatography on a Chiralpak AD (20 mm x 250 mm) column
eluting with 100% ethanol at a flow rate of 10 mL/min. After combination of fractions
and evaporation of the solvent in vacua, one peak with a retention time of 3.801 minutes
was isolated as a white solid (144 mg, 79% based upon a 1:1 ratio of enantiome with a
theoretical maximum amount of 183.5 mg). [a]D
25 = -398° (c = 0.010 g/mL, CHC13); H
NMR (400 MHz, DMSO-d6) 5 ppm 1.21 (d, .7=6.73 Hz, 3 H) 5.65 (d, .7=6.47 Hz, 1 H)
6.70 (d, .7=7.50 Hz, 1 H) 6.73 (s, 1 H) 6.84 (ddd, J=8.15, 2.46, 0.78 Hz, 1 H) 6.92 (s, 1 H)
7.17 (t, J=7.89 Hz, 1 H) 7.28 (dd, J=8.41, 2.20 Hz, 1 H) 7.39 (m, 2 H) 7.45 (m, 1 H) 7.98
(d, .7=8.54 Hz, 2 H) 9.68 (s, 1 H); MS (ESI) m/z 350/352 ([M+H]+); MS (ESI) m/z
57
348/350 ([M-HD; HRMS: calcd for C2iHi6ClNO2, 349.0870 (M), 350.09424 ([M+H]+);
found (ESI+), 350.09353.
Example 26: Synthesis of 3-{[(6S)-3-chloro-6-methylphenanthridin-5(6//)-
yl]carbonyl} phenol
The enantiome of 3-[(3-chloro-6-methylphenanthridin-5(6//)-
yl)carbonyl]phenol (367 mg, 1.05 mmol) were separated by automated, preparative,
normal phase, chiral chromatography on a Chiralpak AD (20 mm x 250 mm) column
eluting with 100% ethanol at a flow rate of 10 mL/min. After combination of fractions
and evaporation of the solvent in vacua, one peak (99.9%) with a retention time of 9.771
minutes was isolated as a white solid (135 mg, 74% based upon a 1:1 ratio of
enantiome with a theoretical maximum amount of 183.5 mg).
[a]D
25 = +440° (c = 0.010 g/mL, CHC13); 1H NMR (400 MHz, DMSO-d6) 8 ppm 1.21 (d,
.7=6.73 Hz, 3 H) 5.64 (d, .7=6.21 Hz, 1 H) 6.69 (d, .7=7.50 Hz, 1 H) 6.72 (s, 1 H) 6.83 (dd,
.7=2.59, 1.03 Hz, 1 H) 6.85 (m, 1 H) 6.92 (s, 1 H) 7.16 (t, .7=7.89 Hz, 1 H) 7.28 (dd,
.7=8.41, 2.20 Hz, 1 H) 7.39 (m, 2 H) 7.45 (m, 1 H) 7.98 (d, .7=8.54 Hz, 2 H); MS (ESI)
m/z 350/352 ([M+H]+);
MS (ESI) m/z 348/350 ([M-H]'); HRMS: calcd for C2iHi6ClNO2, 349.0870 (M),
350.09424 ([M+H]+); found (ESI+), 350.09345.
Example 27: Synthesis of 4-[(3-chloro-6-methylphenanthridin-5(6//)-
yl)carbonyl]phenol
Step A: N-[ 1 -(4'-chloro-2'-fluoro-1.1 '-biphenvl-2-yl)ethyl]-4-methoxybenzamide
- The title compound was prepared from l-(4'-chloro-2'-fluoro-biphenyl-2-yl)-ethylamine
(1 g, 4 mmol), triethylamine (614 uL, 4.4 mmol), and 4-methoxybenzoyl chloride (683
mg, 4 mmol) according to the procedure and in the same manner as described in Example
24, step c. The crude residue was immediately purified on a Biotage® (40Mi) column
for flash column chromatography (5-30% methyl tert-butylether in hexane) resulting in
the isolation of 1.22 g (79%) of a white solid, mp 165-165.9 °C; 1H NMR (400 MHz,
DMSO-de) 8 ppm 1.31 (s, 3 H) 3.80 (s, 3 H) 4.94 (s, 1 H) 6.97 (d, .7=8.79 Hz, 2 H) 7.15
(d, .7=7.50 Hz, 1 H) 7.30 (s, 1 H) 7.43 (m, 2 H) 7.60 (m, 3 H) 7.82 (d, .7=7.50 Hz, 2 H)
8.66 (s, 1 H). MS (ESI) m/z 384/386 ([M+H]+); Anal, calcd for C22Hi9ClFNO2: C:68.84
H:4.99N:3.65 Found: C:68.81 H:5.07N:3.65.
58
Step B: 3-chloro-5-(4-methoxvbenzovlV6-methvl-5.6-dihydrophenanthridine -
The title compound was prepared from N-[l-(4'-chloro-2'-fluoro-biphenyl-2-yl)-ethyl]-4-
methoxy-benzamide (1.04 g, 2.7 mmol), anhydrous THF (10 mL), and lithium
bis(trimethylsilyl)amide (4.1 mL, IM in THF) according to the procedure and in the same
manner as described in Example 24, step d. The THF was removed and the resulting
residue was purified by flash column chromatography (Biotage® 40 Mi, 30-50% methyl
tert-butylether in hexane) affording 738 mg (75%) of the desired product. MS [(ES+),
m/z]: 364/366 [M+H]+, 1 Cl pattern;
Step C: 4- [(3 -chloro-6-methylphenanthridin-5 (6H)-vllcarbonyll phenol - The title
compound was prepared from (3-chloro-6-methyl-6H-phenanthridin-5-yl)-(4-methoxyphenyl)-
methanone (622 mg, 1.71 mmol), cyclohexene (900 uL, 8.9 mmol), and 1 M
solution of boron tribromide in dichloromethane (6.8 mL) according to the procedure and
in the same manner as described in Example 24, step e. The crude residue was
immediately purified using a Biotage® 40 Mi column of prepacked silica gel (90g),
eluting with a gradient of 5-30% methyl terf-butylether in hexane at a flow rate of 50
mL/min. The fractions containing the desired product were combined and concentrated to
afford 450 mg (75%) of a white solid. !H NMR (400 MHz, DMSO-d6) 6 ppm 1.21 (d,
J=6.73 Hz, 3 H) 5.67 (q, J=6.73 Hz, 1 H) 6.71 (d, .7=8.79 Hz, 2 H) 6.79 (d, .7=2.07 Hz, 1
H) 7.17 (d, .7=8.54 Hz, 2 H) 7.26 (dd, .7=8.54, 2.07 Hz, 1 H) 7.41 (m, 3 H) 7.98 (m, 2 H)
10.03 (s, 1 H); MS (ESI) m/z 350/352 ([M+H]+); MS (ESI) m/z 348/350 ([M-H]").
Example 28: Synthesis of 4-{[(6)-3-chloro-6-methylphenanthridin-5(6//)-
yl]carbonyl}phenol
The enantiome of 4-[(3-chloro-6-methylphenanthridin-5(67/)-
yl)carbonyl]phenol (400 mg, 1.14 mmol) were separated by automated, on-column
solvent change, preparative, normal phase, chiral chromatography on a Chiralpak AD (20
mm x 250 mm) column eluting with 20% ethanol in hexane at a flow rate of 12 mL/min
with. After combination of fractions and evaporation of the solvent in vacua, one peak
(99.9%) with a retention time of 3.571 minutes was isolated as a white solid (194 mg,
97% based upon a 1:1 ratio of enantiome with a theoretical maximum amount of 200
mg). mp 273.5-276 °C; [a]D
25 = -321° (c = 0.010 g/mL, CHC13); H NMR (400 MHz,
59
DMSO-de) 6 ppm 1.21 (d, .7=6.73 Hz, 3 H) 5.67 (q, J=6.90 Hz, 1 H) 6.71 (d, J=8.79 Hz,
2 H) 6.79 (d, J=2.07 Hz, 1 H) 7.17 (m, 2 H) 7.26 (dd, .7=8.54, 2.07 Hz, 1 H) 7.41 (m, 3
H) 7.98 (dd, 2 H) 10.04 (s, 1 H); MS (ESI) m/z 350/352 ([M+H]+); MS (ESI) m/z 348/350
([M-H]').
Example 29: Synthesis of 4-{[(6iS)-3-chloro-6-methylphenanthridin-5(6//)-
y l]carbony 1} phenol
The enantiome of 4-[(3-chloro-6-methylphenanthridin-5(6//)-yl)carbonyl]phenol (400
mg, 1.14 mmol) were separated by automated, on-column solvent change, preparative,
normal phase, chiral chromatography on a Chiralpak AD (20 mm x 250 mm) column
eluting with 20% ethanol in hexane at a flow rate of 12 mL/min with. After combination
of fractions and evaporation of the solvent in vacua, one peak (99.8%) with a retention
time of 8.078 minutes was isolated as a white solid (200 mg, 100% based upon a 1:1 ratio
of enantiome with a theoretical maximum amount of 200 mg). mp 273.7-276 °C; [a]o25
= +393° (c = 0.010 g/mL, CHC13); !H NMR (400 MHz, DMSO-d6) 8 ppm 1.21 (d,
7=6.73 Hz, 3 H) 5.67 (q, .7=6.73 Hz, 1 H) 6.71 (d, .7=8.79 Hz, 2 H) 6.79 (d, .7=2.07 Hz, 1
H) 7.17 (d, J=8.54 Hz, 2 H) 7.26 (dd, J=8.41, 2.20 Hz, 1 H) 7.41 (m, 3 H) 7.98 (m, 2 H)
10.04 (s, 1 H); MS (ESI) m/z 350/352 ([M+H]+);
MS (ESI) m/z 348/350 ([M-H]').
Example 30: Synthesis of 4-[3-chloro-6-methylphenanthridin-5(6//)-
yl]carbonyl]benzene-l ,3-diol.
Step A: N-[l -(4'-chloro-2'-fluoro-1.1 '-biphenvl-2-vnethvl1-2.4-
dimethoxvbenzamide - The title compound was prepared from l-(4'-chloro-2'-fluorobiphenyl-
2-yl)-ethylamine (500 mg, 2 mmol), triethylamine (293 uL, 2.1 mmol), and 2,4-
dimethoxybenzoyl chloride (342 mg, 2 mmol) according to the procedure and in the same
manner as described in Example 24, step c. The acetonitrile was removed in vacua and
the resulting solid dissolved in dichloromethane and applied directly to a Biotage®
(40Mi) column for flash column chromatography (5-30% methyl tert-butylether in
hexane) resulting in the isolation of 820 mg (99%) of a white solid.
MS (ESI) m/z 414/416 ([M+H]+.
60
Step B: 3-chloro-5-(2.4-dimethoxvbenzovl)-6-methvl-5.6-dihydrophenanthridine
- The title compound was prepared from N-[l-(4'-chloro-2'-fluoro-biphenyl-2-yl)-ethyl]-
4-methoxy-benzamide (800 mg, 1.9 mmol), anhydrous THF (10 mL), and lithium
bis(trimethylsilyl)amide (3.8 mL, IM in THF) according to the procedure and in the same
manner as described in Example 24, step d. The THF was removed and the resulting
residue was purified by flash column chromatography (Biotage® 40 Mi, 30-50% methyl
tert-butylether in hexane) affording 564 mg (75%) of the desired product. MS [(ES+),
m/z]: 394/396 [M+H]+, 1 Cl pattern;
Step C: 4-[3-chloro-6-methvlphenanthridin-5(6//)-vl1carbonyl]benzene-1.3-diol -
The title compound was prepared from (3-chloro-6-methyl-6H-phenanthridin-5-yl)-(4-
methoxy-phenyl)-methanone (564 mg, 1.43 mmol), cyclohexene (1.45 mL, 14.5 mmol),
and 1 M solution of boron tribromide in dichloromethane (8.59 mL) according to the
procedure and in the same manner as described in Example 1, step e. The resulting
residue was immediately purified using a Biotage® 40 Mi column of prepacked silica gel
(90g), eluting with a gradient of 5-50% methyl tert-butylether in hexane at a flow rate of
50 mL/min. The fractions containing the desired product were combined and
concentrated to afford 516 mg (98%) of an oil. MS [(ES+), m/z]: 366/368 [M+H]+, 1 Cl
pattern; MS [(ESI-) m/z]: 364/366 ([M-H]0, 1 Cl pattern.
Example 31: Synthesis of 4-{[(6)-3-chloro-6-methylphenanthridin-5(6/f)-
yl]carbonyl} benzene-1,3-diol
The enantiome of 4-[3-chloro-6-methylphenanthridin-5(6//)-
yl]carbonyl]benzene-l,3-diol (410 mg, 1.12 mmol) were separated by automated,
preparative, normal phase, chiral chromatography on a Chiralpak AS (20 mm x 250 mm)
column eluting with 15%isopropyl alcohol in hexane at a flow rate of 20 mL/min. After
combination of fractions and evaporation of the solvent in vacuo, one peak (99.9%) with
a retention time of 7.479 minutes was isolated as a white solid (114 mg, 28% based upon
a 1:1 ratio of enantiome with a theoretical maximum amount of 205 mg). [a]o25 = -
367.6° (c = 0.0101 g/mL, CHC13); 'H NMR (400 MHz, DMSO-d6) 8 ppm 1.16 (d, ,7=6.98
Hz, 3 H) 5.64 (d, .7=6.73 Hz, 1 H) 6.16 (d, .7=1.81 Hz, 1 H) 6.23 (dd, .7=8.41, 2.20 Hz,
H) 6.99 (d, .7=8.02 Hz, 2 H) 7.23 (dd, .7=8.54, 2.07 Hz, 1 H) 7.35 (m, 2 H) 7.40 (m, 1 H)
61
7.92 (m, 2 H) 9.60 (s, 1 H) 9.69 (s, 1 H); MS (ESI) m/z 366/368 ([M+H]+);
MS (ESI) m/z 364/366 ([M-H]'); HRMS: calcd for C2iHi6ClNO3, 365.0819 (M),
366.08915 ([M+H]+); found (ESI+), 366.08918.
Example 32: Synthesis of 4-{[(6S)-3-chloro-6-methylphenanthridin-5(6//)-
yl]carbonyl} benzene-1,3-diol
The enantiome of 4-[3-chloro-6-methylphenanthridin-5(6//)-
yl]carbonyl]benzene-l,3-diol (410 mg, 1.12 mmol) were separated by automated,
preparative, normal phase, chiral chromatography on a Chiralpak AS (20 mm x 250 mm)
column eluting with 15%isopropyl alcohol in hexane at a flow rate of 20 mL/min. After
combination of fractions and evaporation of the solvent in vacua, one peak (99.6%) with
a retention time of 9.360 minutes was isolated as a white solid (115 mg, 28% based upon
a 1:1 ratio of enantiome with a theoretical maximum amount of 205 mg). [a]o25 =
+359.5° (c = 0.010 g/mL, CHC13); !H NMR (400 MHz, DMSO-d6) 6 ppm 1.16 (d, J=6.73
Hz, 3 H) 5.64 (d, J=6.47 Hz, 1 H) 6.16 (d, J=1.55 Hz, 1 H) 6.23 (dd, J=8.28, 2.33 Hz, 1
H) 6.99 (d, .7=8.28 Hz, 2 H) 7.23 (dd, J=8.54, 2.07 Hz, 1 H) 7.35 (m, 2 H) 7.40 (m, 2 H)
7.92 (dd, 2 H) 9.60 (s, 1 H) 9.69 (s, 1 H); MS (ESI) m/z 366/368 ([M+H]+);
MS (ESI) m/z 364/366 ([M-H]'); HRMS: calcd for C2iHi6ClNO3, 365.0819 (M),
366.08915 ([M+H]+); found (ESI+), 366.0892.
Example 33: Synthesis of 3-[(3-fluoro-6-methylphenanthridin-5(6//)-
yl)carbonyl]phenol
Step A: 1 -(2'.4'-difluoro-1.1 '-biphenvl-2-vnethanone - 2,4-Difluorophenyl
boronic acid (9.47 g, 60 mmol), tetrabutylammonium bromide (16.1 g, 50 mmol), and
potassium carbonate (20.7 g, 150 mmol) were added to a flask followed by water (50
mL). The contents were mixed until most of the dissolvable solids were in solution. To
the remaining slurry was added 2'-bromoacetophenone (9.95 g, 50 mmol) and palladium
acetate (1.12 g, 5 mmol). The flask was equipped with a condenser and the contents were
heated (70 °C) with stirring while the system was immeed with a nitrogen atmosphere.
After 12 h, TLC analysis indicated the formation of a single product at the expense of 2'-
bromoacetophenone. Heating was discontinued and the reaction was allowed to cool.
62
Ethyl acetate (500 mL) was added and the organic phase was extracted with water (3 x
100 mL). The combined water phase was extracted once with additional ethyl acetate
and the organic phases were combined, dried (NaaSC), filtered and concentrated to
provide a crude oil. The product was isolated via flash column chromatography
(Biotage® 40 Mi, 5-20%, methyl tert-bulyl ether in hexane) and afforded 5.7 g (49%) of
the desired product as a yellow oil. MS (El) m/z 233 (M+.); H NMR (400 MHz, DMSOd6)
8 ppm 2.42 (s, 3 H) 7.16 (m, 1 H) 7.27 (ddd, .7=10.57, 9.29, 2.43 Hz, 1 H) 7.39 (m, 2
H) 7.56 (td, .7=7.56, 1.54 Hz, 1 H) 7.64 (td, .7=7.49, 1.41 Hz, 1 H) 7.86 (dd, .7=7.43, 1.28
Hz, 1 H).
Step B;l-(-2'.4'-difluoro-biphenvl-2-ylVethvlamine - The title compound was
prepared from l-(2',4'-difluoro-l,r-biphenyl-2-yl)ethanone (3.67 g, 15.8 mmol),
anhydrous methanol (200 mL), ammonium acetate (12.2 g, 158 mmol), and sodium
cyanoborohydride (1.99 g, 31.6 mmol) according to the procedure and in the same
manner as described in Example 24, step b resulting in the isolation of a clear oil that was
used without further purification. MS [(ES+), m/z]: 217 [M-16]+, benzylic cation as a
result of the loss of NH2-
Step C: A-ri-(2'.4'-difluoro-l.r-biphenvl-2-vnethvll-3-methoxvbenzamide - 3-
Methoxybenzoyl chloride (731 mg, 4.28 mmol) was added to a solution of l-(-2',4'-
difluoro-biphenyl-2-yl)-ethylamine (1.0 g, 4.28 mmol) and triethylamine (717 uL, 5.1
mmol) in dichloromethane (lOmL). The reaction was mixed overnight (16 h) on an
orbital mixer at room temperature. The volume of dichloromethane was reduced in
vacua by 50% and the reaction contents were applied directly to a Biotage® (40Mi)
column for flash column chromatography (5-30% methyl tert-butylether in hexane)
resulting in the isolation of 1.52 g (97%) of a white solid, mp 163.9-165 °C; 'H NMR
(400 MHz, DMSO-de) 5 ppm 1.30 (br d, 7=6.21 Hz, 3 H) 3.79 (s, 3 H) 4.96 (br m, 1 H)
7.07 (dd, J=8.02, 1.81 Hz, 1 H) 7.15 (d, .7=7.50 Hz, 2 H) 7.32 (m, 4 H) 7.44 (m, 2 H) 7.63
(s, 2 H) 8.79 (s, 1 H); MS (ESI) m/z 368 ([M+H]+); Anal, calcd for C22Hi9F2NO2:
C:71.92 H:5.21 N:3.81 Found: C:71.54 H:5.27 N:3.73
Step D: 3-fluoro-5-(3-rnethoxvbenzovl)-6-methvl-5.6-dihydrophenanthridine -
A-[l-(2',4'-difluoro-l,r-biphenyl-2-yl)ethyl]-3-methoxybenzamide (1.29 g, 3.5 mmol)
was dissolved in anhydrous THF (10 mL) and the vial was capped and purged with an
inert atmosphere. Lithium bis(trimethylsilyl)amide (4.0 mL, 1M in THF) was added to
the solution and the mixture heated (70 °C). The reaction progress was monitored by
LCMS and heating was discontinued upon the expense of the starting material. The THF
was removed and 2N aqueous HC1 was added. The product was extracted with ethyl
acetate (3x) and the combined organic phases were dried (Na2S04), filtered, and
concentrated to provide a crude oil. The residue was purified by flash column
chromatography (Biotage® 40 Mi, 30-50% methyl tert-butylether in hexane) affording
1.0 g (82%) of the desired product. H NMR (400 MHz, DMSO-d6) S ppm 1.22 (d,
.7=6.76 Hz, 3 H) 3.71 (s, 3 H) 5.67 (s, 1 H) 6.77 (d, .7=7.28 Hz, 2 H) 6.93 (s, 1 H) 7.03
(ddd, .7=8.32, 2.73, 0.91 Hz, 1 H) 7.10 (td, .7=8.71,2.60 Hz, 1 H) 7.26 (t, .7=7.93 Hz, 1 H)
7.40 (m, 3 H) 7.97 (d, .7=7.54 Hz, 1 H) 8.01 (dd, J=8.84, 6.24 Hz, 1 H); MS (ESI) m/z
348 ([M+H]+);
HRMS: calcd for C22Hi8FN02, 347.1322 (M), 348.13944 ([M+H]+); found (ESI_FT),
348.13903; Anal. Calcd for C22Hi8FNO2: C, 76.07; H, 5.22; N, 4.03. Found: C, 76.02; H,
5.32; N, 3.97.
Step E: 3-[(3-fluoro-6-methvlphenanthridin-5(6//)-vncarbonyl]phenol - To a
solution of 3-fluoro-5-(3-methoxybenzoyl)-6-methyl-5,6-dihydrophenanthridine (878 mg,
2.5 mmol) in dichloromethane was added cyclohexene (1.0 mL, 10.1 mmol). The vial
was capped and purged with an inert atmosphere followed by the addition of a 1 M
solution of boron tribromide in dichloromethane (10 mL). The solution was mixed at
room temperature using an orbital shaker. After 3 hou the reaction was cooled in an ice
bath (0 °C) and quenched upon the slow addition of methanol. The clear solution was
concentrated and the resulting residue was immediately purified using a Biotage® 40 Mi
column of prepacked silica gel (90g), eluting with a gradient of 5-30% methyl tertbutylether
in hexane at a flow rate of 50 mL/min. The fractions containing the desired
product were combined and concentrated to afford 731 mg (85%) of a white solid.
'H NMR (400 MHz, DMSO-d6) 8 ppm 1.21 (d, .7=6.73 Hz, 3 H) 5.66 (q, 1 H) 6.71 (m, 3
H) 6.84 (ddd, .7=8.21, 2.52, 0.91 Hz, 1 H) 7.09 (td, J=8.54, 2.59 Hz, 1 H) 7.16 (t, .7=7.76
Hz, 1 H) 7.37 (m, 2 H) 7.44 (td, .7=7.37, 1.81 Hz, 1 H) 7.97 (d, .7=7.50 Hz, 1 H) 8.00 (dd,
.7=8.79, 6.21 Hz, 1 H) 9.66 (s, 1 H); MS (ESI) m/z 334 ([M+H]+); MS (ESI) m/z 332 ([M-
64
Example 34: Synthesis of 3-{[(6/?)-3-fluoro-6-methylphenanthridin-5(6//)-
yl]carbonyl}phenol
The enantiome of 3-[(3-fluoro-6-methylphenanthridin-5(6//)-
yl)carbonyl]phenol (660 mg, 1.98 mmol) were separated by automated, preparative,
normal phase, chiral chromatography on a Chiralpak AD (20 mm x 250 mm) column
eluting with 100% acetonitrile at a flow rate of 20 mL/min with. The fractions containing
the fit peak were combined and concentrated in vacuo, to provide one peak (99.9%)
with a retention time of 2.708 minutes was isolated as a white solid (270 mg, 82% based
upon a 1:1 ratio of enantiome with a theoretical maximum amount of 330 mg). [a]o25 =
-545° (c = 0.0105 g/mL, CHC13); 'H NMR (400 MHz, DMSO-d6) 8 ppm 1.21 (d, .7=6.98
Hz, 3 H) 5.66 (q, /=6.38 Hz, 1 H) 6.69 (d, .7=7.24 Hz, 2 H) 6.72 (s, 1 H) 6.83 (ddd,
.7=8.15, 2.46, 0.78 Hz, 1 H) 7.09 (td, .7=8.67, 2.59 Hz, 1 H) 7.16 (t, .7=7.89 Hz, 1 H)
(m, 2 H) 7.44 (td, 1 H) 7.96 (d, .7=7.76 Hz, 1 H) 8.00 (dd, .7=8.79, 6.47 Hz, 1 H) 9.66 (s,
H) MS (ESI) m/z 334 ([M+Hf); MS (ESI) m/z 332 ([M-H]"); HRMS: calcd for
C2iHi6FNO2, 333.1165 (M), 334.12379 ([M+Hf); found (ESI+), 334.12345.
Example 35: Synthesis of 3-{[(65)-3-fluoro-6-methylphenanthridin-5(67/)-
yl]carbonyl}phenol
The enantiome of 3-[(3-fluoro-6-methylphenanthridin-5(6//)-
yl)carbonyl]phenol (660 mg, 1.98 mmol) were separated by automated, preparative,
normal phase, chiral chromatography on a Chiralpak AD (20 mm x 250 mm) column
eluting with 100% acetonitrile at a flow rate of 20 mL/min with. The fractions containing
the second peak were combined and concentrated in vacuo, affording a white solid (270
mg, 82% based upon a 1:1 ratio of enantiome with a theoretical maximum amount of
330 mg one peak (99.9%) with a retention time of 3.894 minutes; [a]D
25 = +490° (c =
0.0102 g/mL, CHC13); 'H NMR (400 MHz, DMSO-d6) 6 ppm 1.21 (d, .7=6.98 Hz, 3 H)
5.66 (q, 1 H) 6.69 (d, .7=7.50 Hz, 2 H) 6.72 (s, 1 H) 6.83 (ddd, .7=8.15, 2.46, 1.03 Hz, 1
H) 7.09 (m, 1 H) 7.16 (t, .7=7.89 Hz, 1 H) 7.37 (m, 2 H) 7.44 (m, .7=7.37, 7.37,1.81 Hz,
H) 7.96 (d, .7=7.50 Hz, 1 H) 8.00 (dd, 7=8.79, 6.21 Hz, 1 H) 9.66 (s, 1 H); MS (ESI) m/z
334 ([M+H]+); MS (ESI) m/z 332 ([M-H]'); HRMS: calcd for C2iH16FNO2, 333.1165
(M), 334.12379 ([M+Hf); found (ESI+), 334.12344.
Example 36: Synthesis of 4-[(3-fluoro-6-methylphenanthridin-5(6//)-
yl)carbonyl]phenol
Step A: N-\\ -(2'.4'-difluoro- 1 . 1 '-biphenvl-2-vnethvl1-4-methoxvbenzamide - The
title compound was prepared from 4-methoxybenzoyl chloride (731 mg, 4.28 mmol), l-(-
2',4'-difluoro-biphenyl-2-yl)-ethylamine (1 g, 4.28 mmol), and triethylamine (717 uL, 5.1
mmol) according to the procedure and in the same manner as described in Example 33,
step c. The product was isolated from a Biotage® (40Mi) column for flash column
chromatography (5-30% methyl tert-butylether in hexane) resulting in the isolation of a
white solid, 1.53 g (97%).
mp 134.5-135 °C; 'H NMR (400 MHz, DMSO-d6) 8 ppm 1.29 (d, .7=5.43 Hz, 3 H) 3.80
(s, 3 H) 4.96 (m, .7=5.95 Hz, 1 H) 6.97 (d, .7=9.05 Hz, 2 H) 7.18 (m, 2 H) 7.36 (m, 3 H)
7.64 (d, .7=4.14 Hz, 2 H) 7.83 (d, J=7.50 Hz, 2 H) 8.66 (s, 1 H); MS (ESI) m/z 368
Anal, calcd for C22Hi9F2NO2: C:71.92 H:5.21 N:3.81 Found: C:72.11 H:5.23 N:3.77
Step B: 3-fluoro-5-(4-methoxvbenzovn-6-methvl-5.6-dihvdrophenanthridine -
The title compound was prepared from W-[l-(2',4'-difluoro-l,r-biphenyl-2-yl)ethyl]-4-
methoxybenzamide (1.29 g, 3.5 mmol), anhydrous THF (10 mL), and lithium
bis(trimethylsilyl)amide (4.0 mL, 1M in THF) according to the procedure and in the same
manner as described in Example 33, step d. The residue was purified by flash column
chromatography (Biotage® 40 Mi, 30-50% methyl tert-butylether in hexane) affording
1.1 g (90%) of the desired product. 1H NMR (400 MHz, DMSO-d6) 8 ppm 1.21 (d,
J=6.76 Hz, 3 H) 3.77 (s, 3 H) 5.68 (q, 7=6.67 Hz, 1 H) 6.60 (dd, 7=10.39, 2.60 Hz, 1 H)
6.90 (d, .7=8.84 Hz, 2 H) 7.07 (td, .7=8.64, 2.73 Hz, 1 H) 7.27 (d, .7=8.58 Hz, 2 H) 7.40
(m, 3 H) 7.97 (m, 1 H) 8.00 (dd, .7=8.84, 6.24 Hz, 1 H); MS (ESI) m/z 348 ([M+H]+);
HRMS: calcd for C22Hi8FN02, 347.1322 (M), 348.13944 ([M+H]+); found (ESI_FT),
348.13914.
Step C: 4-[(3-fluoro-6-methvlphenanthridin-5(6//)-yncarbonyl]phenol -_The title
compound was prepared 3-fiuoro-5-(4-methoxybenzoyl)-6-methyl-5,6-
dihydrophenanthridine (977 mg, 2.8 mmol), cyclohexene (1.2 mL, 11.3 mmol), and 1 M
solution of boron tribromide in dichloromethane (1 1 .3 mL) according to the procedure
and in the same manner as described in Example 33, step e. The crude residue was
immediately purified using a Biotage® 40 Mi column of prepacked silica gel (90g),
eluting with a gradient of 5-30% methyl tert-butylether in hexane at a flow rate of 50
mL/min. The fractions containing the desired product were combined and concentrated to
afford 773 mg (90%) of a white solid.
'H NMR (400 MHz, DMSO-d6) 8 ppm 1.21 (d, .7=6.98 Hz, 3 H) 5.67 (q, .7=6.73 Hz, 1 H)
6.56 (dd, .7=10.60, 2.59 Hz, 1 H) 6.70 (d, .7=8.79 Hz, 2 H) 7.06 (td, .7=8.54, 2.59 Hz, 1 H)
7.16 (d, .7=8.28 Hz, 2 H) 7.39 (m, 3 H) 7.96 (d, .7=7.76 Hz, 1 H) 7.99 (dd, J=8.92, 6.34
Hz, 1 H) 10.03 (s, 1 H); MS (ESI) m/z 334 ([M+H]+); MS (ESI) m/z 332 ([M-H]').
Example 37: Synthesis of 4-{[(6#)-3-fluoro-6-methylphenanthridin-5(6/f)-
yljcarbonyl} phenol
The enantiome of 4-[(3-fluoro-6-methylphenanthridin-5(6/f)-
yl)carbonyl]phenol (720 mg, 2.16 mmol) were separated by automated, on-column
solvent change, preparative, normal phase, chiral chromatography on a Chiralpak AD (20
mm x 250 mm) column eluting with 35% hexane in ethanol at a flow rate of 15 mL/min
with. The fractions containing the fit peak were combined and concentrated in vacua,
to provide one peak (99.9%) with a retention time of 3.620 minutes was isolated as a
white solid (318 mg, 88% based upon a 1:1 ratio of enantiome with a theoretical
maximum amount of 360 mg). mp 235.7-236.8 °C; [a]D
25 = -648° (c = 0.010 g/mL,
CHC13); 'H NMR (400 MHz, DMSO-d6) 5 ppm 1.21 (d, .7=6.98 Hz, 3 H) 5.68 (q, .7=6.73
Hz, 1 H) 6.56 (dd, .7=10.48, 2.46 Hz, 1 H) 6.70 (d, .7=8.79 Hz, 2 H) 7.06 (td, .7=8.60,
Hz, 1 H) 7.16 (m, 2 H) 7.35 (m, 1 H) 7.42 (m, 2 H) 7.98 (td, .7=8.73, 7.11 Hz, 2 H) 10.04
(s, 1 H)
MS (ESI) m/z 334 ([M+H]+); MS (ESI) m/z 332 ([M-H]').
Example 38: Synthesis of 4-{[(6S)-3-fluoro-6-methylphenanthridin-5(67/)-
yl]carbonyl}phenol
The enantiome of 4-[(3-fluoro-6-methylphenanthridin-5(6/f)-
yl)carbonyl]phenol (720 mg, 2.16 mmol) were separated by automated, on-column
solvent change, preparative, normal phase, chiral chromatography on a Chiralpak AD (20
mm x 250 mm) column eluting with 35% hexane in ethanol at a flow rate of 15 mL/min
67
with. The fractions containing the second peak were combined and concentrated in
vacua, to provide one peak (99.8%) with a retention time of 8.095 minutes was isolated
as a white solid (345 mg, 96% based upon a 1:1 ratio of enantiome with a theoretical
maximum amount of 360 mg). mp 236.6-237.8 °C; [a]D
25 = +581° (c = 0.0107 g/mL,
CHC13); 1H NMR (400 MHz, DMSO-d6) 8 ppm 1.21 (d, 7=6.73 Hz, 3 H) 5.68 (q, .7=6.73
Hz, 1 H) 6.56 (dd, .7=10.60, 2.59 Hz, 1 H) 6.70 (d, .7=8.79 Hz, 2 H) 7.06 (td, .7=8.54, 2.59
Hz, 1 H) 7.16 (m, 2 H) 7.35 (m, 1 H) 7.42 (m, 2 H) 7.98 (td, .7=8.79, 6.98 Hz, 2 H) 10.04
(s, 1 H)
MS (ESI) m/z 334 ([M+H]+); MS (ESI) m/z 332 ([M-H]').
Example 39: Synthesis of 4-[(3-fluoro-6-methylphenanthridin-5(6//)-
yl)carbonyl]benzene-1,3-diol
Step A: A-ri-Q''-difluoro-l.r-biphenvl-vllethvll-dimethoxvbenzamide -
The title compound was prepared from 2,4-dimethoxybenzoyl chloride (860 mg, 4.28
mmol), l-(-2',4'-difluoro-biphenyl-2-yl)-ethylamine (1 g, 4.28 mmol), and triethylamine
(717 uL, 5.1 mmol) according to the procedure and in the same manner as described in
Example 33, step c resulting in the isolation of 1.47 g (86%) of a white solid, mp 134.8-
135.2 °C; H NMR (400 MHz, DMSO-de) 5 ppm 1.29 (d, .7=5.95 Hz, 3 H) 3.81 (s, 3 H)
3.92 (s, 3 H) 4.93 (d, .7=6.73 Hz, 1 H) 6.59 (dd, J=8.79,2.33 Hz, 1 H) 6.64 (d, .7=2.33 Hz,
1 H) 7.18 (m, 2 H) 7.49 (m, 6 H) 8.29 (s, 1 H); MS (ESI) m/z 398 ([M+H]+); Anal, calcd
for C23H2iF2NO3: C:69.51 H:5.33 N:3.52 Found: C:69.27 H:5.30 N:3.39
Step B: 3-fluoro-5-(2<4-dimethoxvbenzovlV6-methvl-5.6-dihydrophenanthridine
- The title compound was prepared from Af-[l-(2',4'-difluoro-l,r-biphenyl-2-yl)ethyl]-
2,4-dimethoxybenzamide (1.29 g, 3.2 mmol), anhydrous THF (10 mL), and lithium
bis(trimethylsilyl)amide (4.0 mL, 1M in THF) according to the procedure and in the same
manner as described in Example 33, step d. The resulting residue was purified by flash
column chromatography (Biotage® 40 Mi, 30-50% methyl tert-butylether in hexane)
affording 1.2 g (99%) of the desired product. 'H NMR (400 MHz, DMSO-d6) 5 ppm 1.16
(d, .7=6.76 Hz, 3 H) 3.79 (br m, 6 H) 3.92 (s, 1 H) 6.28 (s, 1 H) 6.62 (m, 1 H) 7.06 (s, 1
H) 7.34 (br m, 1 H) 7.42 (br m, 3 H) 7.52 (br d, .7=7.54 Hz, 1 H) 7.96 (m, 2 H); MS (ESI)
m/z 378 ([M+H]+);
HRMS: calcd for C23H2oFNO3, 377.1427 (M), 378.15000 ([M+H]+); found (ESI_FT),
378.14878
Step C: 4-[(3-fluoro-6-methvlphenanthridin-5(6//)-vl)carbonvl1benzene-l,3-diol
The title compound was 3-fluoro-5-(2,4-dimethoxybenzoyl)-6-methyl-5,6-
dihydrophenanthridine (1.25 mg, 3.3 mmol), cyclohexene (1.74 mL, 16.5 mmol), and 1
M solution of boron tribromide in dichloromethane (16.5 mL) according to the procedure
and in the same manner as described in Example 33, step e. The crude residue was
immediately purified using a Biotage® 40 Mi column of prepacked silica gel (90g),
eluting with a gradient of 5-30% methyl terf-butylether in hexane at a flow rate of 50
mL/min. The fractions containing the desired product were combined and concentrated to
afford 1.0 g (85%) of a white solid. 'H NMR (400 MHz, DMSO-d6) 8 ppm 1.16 (d,
.7=6.98 Hz, 3 H) 5.65 (d, .7=6.73 Hz, 1 H) 6.16 (d, .7=1.81 Hz, 1 H) 6.22 (dd, .7=8.41, 2.20
Hz, 1 H) 6.75 (d, .7=9.83 Hz, 1 H) 6.98 (d, .7=8.28 Hz, 1 H) 7.04 (td, J=8.60, 2.72 Hz, 1
H) 7.36 (m, 3 H) 7.90 (d, .7=7.50 Hz, 1 H) 7.94 (dd, 7=8.92, 6.34 Hz, 1 H) 9.62 (s, 1 H)
9.70 (s, 1 H)
MS (ESI) m/z 350 ([M+H]+); MS (ESI) m/z 348 ([M-H]'); HRMS: calcd for C2iHi6FNO3,
349.1114 (M), 348.10414 ([M+H]+); found (ESI-), 348.10418.
Example 40: Synthesis of 4-{[(6/J)-3-fluoro-6-methylphenanthridin-5(6//)-
yl]carbonyl}benzene-l ,3-diol
The enantiome of 4-[(3-fluoro-6-methylphenanthridin-5(6//)-
yl)carbonyl]benzene-l,3-diol (800 mg, 2.29 mmol) were separated by automated,
preparative, normal phase, chiral chromatography on a Chiralpak AS (20 mm x 250 mm)
column eluting with 15% isopropyl alcohol in hexane at a flow rate of 20 mL/min with.
The fractions containing the fit peak were combined and concentrated in vacua, to
provide one peak (99.9%) with a retention time of 7.971 minutes was isolated as a white
solid (224 mg, 56% based upon a 1:1 ratio of enantiome with a theoretical maximum
amount of 400 mg). mp 184-187 °C; [a]D
25 = -665° (c = 0.0104 g/mL, CHC13); 1H NMR
(400 MHz, DMSO-de) 8 ppm 1.16 (d, 7=6.73 Hz, 3 H) 5.65 (m, 7=6.73 Hz, 1 H) 6.16 (d,
7=1.81 Hz, 1 H) 6.22 (dd, 7=8.41, 2.20 Hz, 1 H) 6.75 (d, 7=9.83 Hz, 1 H) 6.98 (d, 7=8.28
Hz, 1 H) 7.04 (td, 7=8.67, 2.59 Hz, 1 H) 7.33 (m, 2 H) 7.39 (m, 1 H) 7.90 (d, 7=7.76 Hz,
69
1 H) 7.94 (dd, .7=8.79, 6.21 Hz, 1 H) 9.62 (s, 1 H) 9.70 (s, 1 H); MS (ESI) m/z 350
MS (ESI) m/z 348 ([M-H]); HRMS: calcd for C2iHi6FNO3, 349.1114 (M), 350.1187
([M+H]+); found (ESI+), 350.1 181.
Example 41: Synthesis of 4-{[(6S)-3-fluoro-6-methylphenanthridin-5(6//)-
yljcarbonyl } benzene- 1 , 3 -diol
The enantiome of 4-[(3-fluoro-6-methylphenanthridin-5(6//>
yl)carbonyl]benzene- 1,3 -diol (800 mg, 2.29 mmol) were separated by automated,
preparative, normal phase, chiral chromatography on a Chiralpak AS (20 mm x 250 mm)
column eluting with 15% isopropyl alcohol in hexane at a flow rate of 20 mL/min with.
The fractions containing the second peak were combined and concentrated in vacua, to
provide one peak (99.3%) with a retention time of 9.345 minutes was isolated as a white
solid (220 mg, 55% based upon a 1:1 ratio of enantiome with a theoretical maximum
amount of 400 mg). mp 182-184 °C; [a]D
25 = +618° (c = 0.0101 g/mL, CHC13); 1H NMR
(400 MHz, DMSO-de) 6 ppm 1.16 (d, .7=6.73 Hz, 3 H) 5.65 (d, .7=6.73 Hz, 1 H) 6.16 (d,
.7=1.81 Hz, 1 H) 6.22 (dd, .7=8.41, 2.20 Hz, 1 H) 6.75 (d, .7=10.35 Hz, 1 H) 6.98 (d,
.7=8.28 Hz, 1 H) 7.04 (td, .7=8.60, 2.72 Hz, 1 H) 7.33 (m, 2 H) 7.39 (ddd, .7=7.70, 6.79,
2.07 Hz, 1 H) 7.90 (d, .7=7.50 Hz, 1 H) 7.94 (dd, .7=8.79, 6.47 Hz, 1 H) 9.62 (s, 1 H)
MS (ESI) m/z 350 ([M+H]+); MS (ESI) m/z 348 ([M-H]-); HRMS: calcd for C2iHi6FNO3,
349.1 1 14 (M), 350.1 187 ([M+H]+); found (ESI+), 350.1 1817.
Example 42: Synthesis of 4-[(3,8-difluoro-6-methylphenanthridin-5(6#)-
yl)carbonyl]phenol
Step A: 4-Methoxv-./V- f 1 -(2'.4.4'-trifluoro- 1 . 1 '-biphenvl-2-vnethvllbenzamide - A
stirred solution of l-(2',4,4'-trifluoro-l,r-biphenyl-2-yl)ethylamine (0.71 g, 2.84 mmol)
in dichloromethane (5 mL) was treated with 4-methoxybenzoyl chloride (0.51 g, 3.0
mmol), and TV.JV-diisopropylethylamine (0.77 g, 6.0 mmol). The reaction was stirred at
room temperature for twelve hou, and the solvent evaporated in vacuo to a crude oil.
The crude oil was purified by preparative liquid chromatography on a Biotage® 40 Mi
70
column of pre-packed silica gel (90 g), eluting with a gradient of between 5% and 50%
methyl tert-butyl ether in hexane at a flow rate of 40 mL/min to afford, after evaporation
of the solvent, a colorless oil. Crystallization of the colorless oil from ethyl acetatehexane
yielded the title compound (0.98 g, 2.54 mmol, 90%) as a homogeneous,
colorless, crystalline solid, m.p. 163-165 °C; MS [(+ESI), m/z]: 386 [M+H]+;
IR (Solid), vmax: 3257, 1620, 1606,1503, 1329, 1249,1175,1032, 844, 777, 670 cm'1;
'H NMR (400 MHz, DMSO-ok) 5: 1.29 (d, J = 5.4 Hz, 3H), 3.80 (s, 3H), 4.93 (broad s,
•1H), 6.93 (d, J= 8.8 Hz, 2H), 7.14 (td, J= 8.5, 2.7 Hz, IH), 7.22 (dd, J = 8.5, 5.9 Hz,
2H), 7.37 (t, 2H), 7.46 (d, J= 9.1 Hz, IH), 7.61 (d, J= 4.7 Hz, IH), 7.83 (d, J= 7.2 Hz,
2H), 8.66 (d, J= 3.9 Hz, IH), exists as approximate 2:1 mixture of rotame; Anal, calcd
for C22Hi8F3N02: C, 68.57; H, 4.71; N, 3.63. Found: C, 68.65; H, 4.80 N, 3.48.
Step B: 3.8-Difluoro-5-(4-methoxybenzoyl)-6-methyl-5.6-dihvdrophenanthridine
A stirred solution of 4-methoxy-jV-[l-(2',4,4'-trifluoro-l,r-biphenyl-2-
yl)ethyl]benzamide (0.95 g, 2.46 mmol) in tetrahydrofuran was treated under nitrogen
with a 1.0 M solution of lithium bis(trimethylsilyl) amide in tetrahydrofuran (5 mL, 5.0
mmol) and heated at 70 °C for fifteen hou. The reaction mixture was cooled to room
temperature and the solvent removed in vacua to afford a residue. The residue was
dissolved in ethyl acetate and washed sequentially with a 1 N hydrochloric acid solution
and water. The organic phase was dried over anhydrous sodium sulfate, filtered, and the
solvent removed in vacua to afford a crude solid. The crude solid was purified by
preparative liquid chromatography on a Biotage® 40 Mi column of pre-packed silica gel
(90 g), eluting with a gradient of between 3% and 15% methyl tert-butyl ether in hexane
to afford, after crystallization from ethyl acetate-hexane, the title compound (0.36 g, 0.99
mmol, 40%) as a homogeneous, colorless, crystalline solid, m.p. 98-100 °C; MS [(+ESI),
m/z]: 366 [M+Hf; IR (Solid), vmax: 2920, 1630, 1600, 1580, 1510, 1495, 1385, 1320,
1250, 870, 840, 810 cm"1; 'H NMR (400 MHz, DMSO-ak) 8: 1.22 (d,J= 7.0 Hz, 3H),
3.77 (s, 3H), 5.73 (q, J= 6.7 Hz, IH), 6.59 (dd, J= 10.3, 2.3 Hz, IH), 6.90 (d, J = 8.8 Hz,
2H), 7.07 (td, y = 8.6, 2.7 Hz, IH), 7.27 (d, /= 8.3 Hz, 2H), 7.27 (td, J= 8.5, 3.2 Hz,
IH), 7.39 (dd, J= 9.2, 2.7 Hz, IH), 7.98 (dd, J= 8.9, 6.3 Hz, IH), 8.02 (dd, /= 8.5, 5.4
Hz, IH); Anal, calcd for C22Hi7F2NO2: C, 72.32; H, 4.69; N, 3.83. Found: C, 71.82; H,
4.52; N, 3.76.
Step C: 4-[(3.8-Difluoro-6-methvlphenanthridin-5(6/f)-vncarbonvl1phenol - A
stirred suspension of 3,8-difluoro-5-(4-methoxybenzoyl)-6-methyl-5,6-
dihydrophenanthridine (0.20 g, 0.55 mmol) and cyclohexene (1.64 g, 20.0 mmol) was
treated at room temperature under nitrogen with a solution of 1.0 M boron tribromide in
dichloromethane (4.0 mL, 4.0 mmol). After stirring for approximately two hou at room
temperature, the reaction was cooled to -20 °C and quenched with methanol (5 mL). The
solvent was evaporated in vacua to a dark oil. The dark oil was purified by preparative
liquid chromatography on a Biotage® 40 Mi column of pre-packed silica gel (90 g),
eluting with a gradient of between 5% and 30% methyl tert-butyl ether in hexane at a
flow rate of 50 mL/min to afford, after evaporation of the solvent in vacua and trituration
with diethyl ether-hexane, the title compound (0.19 g, 0.54 mmol, 99%) as a
homogeneous, colorless, racemic solid, m.p. 193-195 °C; MS [(+ESI), m/z]: 352 [M+H]+;
MS [(-ESI), m/z]: 350 [M-H]'; 1H NMR (400 MHz, DMSO-rfd) 8: 1.21 (d, J= 7.0 Hz,
3H), 5.71 (q, J= 6.8 Hz, IH), 6.55 (dd, J= 10.5, 2.5 Hz, IH), 6.70 (d, /= 8.6 Hz, 2H),
7.06 (id,J= 8.6, 2.6 Hz, IH), 7.16 (d, J= 8.6 Hz, 2H), 7.27 (td, J= 8.8, 2.7 Hz, IH), 7.38
(dd, J = 9.2, 2.7 Hz, IH), 7.97 (dd, J= 9.0, 6.4 Hz, IH), 8.01 (dd, J= 9.1, 6.0 Hz, IH),
10.04 (s, IH).
Example 43: Synthesis of 4-{[(6)-3,8-Difluoro-6-methylphenanthridin-5(6/f)-
yl] carbonyl} phenol
The enantiome of 4-[(3,8-difluoro-6-methylphenanthridin-5(6/f)-
yl)carbonyl]phenol (0.15 g, 0.43 mmol) were separated by automated, preparative,
normal phase, chiral chromatography on a Chiralpak AD-H® (2 x 25 cm) column eluting
with ethanol at a flow rate of 10 mL/min. After evaporation of the solvent in vacua, peak
one with a retention time at 6.0 minutes and monitored by ultraviolet detection yielded,
after trituration with diethyl ether-hexane, 4-{[(6/?)-3,8-difluoro-6-methylphenanthridin-
5(6/f)-yl]carbonyl}phenol (0.05 g, 0.14 mmol, 33%) as a homogeneous, colorless,
amorphous solid, m.p. 138-140 °C; TR = 6.0 minutes;
25 = -560° (c = 10.0 mg/mL in CHC13); MS [(+ESI), m/z]: 352 [M+H]+; MS [(-ESI),
m/z]: 350 [M-H]'; IR (Solid), v: 3300, 1606, 1570, 1510, 1495, 1390, 1330, 1265,
1225, 870, 810 ran1; H NMR (400 MHz, DMSO-) 6: 1.21 (d, J= 6.8 Hz, 3H), 5.71
(q, J= 6.8 Hz, 1H), 6.55 (dd, J= 10.5, 2.5 Hz, 1H), 6.70 (d, J= 8.8 Hz, 2H), 7.06 (td, J=
8.6, 2.6 Hz, 1H), 7.16 (d, J= 8.6 Hz, 2H), 7.27 (td, .7= 8.8,2.7 Hz, 1H), 7.38 (dd, J= 9.2,
2.7 Hz, 1H), 7.07 (dd, J= 9.0, 6.4 Hz, 1H), 8.01 (dd, J= 9.0, 5.6 Hz, 1H), 10.06 (s, 1H);
Anal, calcd for C2iHi5F2NO2: C, 71.79; H, 4.30; N, 3.99. Found: C, 71.83; H, 4.96; N,
3.54.
The stereochemical configuration is not absolute and was assigned arbitrarily.
Example 44: Synthesis of 4-{[(65)-3,8-Difluoro-6-methylphenanthridin-5(6//)-
yl] carbonyl} phenol
The enantiome of 4-[(3,8-difluoro-6-methylphenanthridin-5(6/f)-
yl)carbonyl]phenol (0.15 g, 0.43 mmol) were separated by automated, preparative,
normal phase, chiral chromatography on a Chiralpak AD-H® (2 x 25 cm) column eluting
with ethanol at a flow rate of 10 mL/min. After evaporation of the solvent in vacua, peak
two with a retention time at 9.5 minutes and monitored by ultraviolet detection yielded,
after trituration with hexane, 4-{[(6S)-3,8-difluoro-6-memylphenanthridin-5(6//)-
yl]carbonyl}phenol (0.04 g, 0.11 mmol, 26%) as a homogeneous, colorless, amorphous
solid, m.p. 135-138 °C; TR = 9.5 minutes; [a]D
25 = +561° (c = 10.0 mg/mL in CHC13);
MS [(+ESI), m/z]: 352 [M+H]+; MS [(-ESI), m/z]: 350 [M-H]';
IR (Solid), Vnm. 3300,1606, 1570, 1510,1495,1390, 1330, 1265, 1225, 870, 810 cm'1;
'HNMR(400 MHz, DMSO-cfc) 8 1.21 (d, J= 6.8 Hz, 3H), 5.71 (q,J= 6.8 Hz, 1H), 6.55
(dd, J= 10.4, 2.6 Hz, 1H), 6.70 (d, J= 8.6 Hz, 2H), 7.06 (td, J = 8.7, 2.6 Hz, 1H), 7.16
(d, J= 8.6 Hz, 2H), 7.27 (td, J= 8.8, 2.7 Hz, 1H), 7.38 (dd, J = 9.1, 2.6 Hz, 1H), 7.97
(dd, J= 8.7, 6.4 Hz, 1H), 8.01 (dd, J = 8.8, 5.5 Hz, 1H), 10.05 (m, 1H); Anal, calcd for
C2iHi5F2N02: C, 71.79; H, 4.30; N, 3.99. Found: C, 70.68; H, 4.50; N, 3.69.
"The stereochemical configuration is not absolute and was assigned arbitrarily.
Example 45: Synthesis of 4-{[(5/2)-3,8-difluoro-6-methylphenanthridin-5((5//)-
yl]carbonyl} -2-fluorophenol
4-{[(5/f)-3,8-difluoro-6-methylphenanthridin-5(5//)-yl]carbonyl}-2-fluorophenol
was prepared in a similar fashion as outlined in example 42. It was purified by chiral
HPLC.
[a]D25 = -453.7° (c = 10 mg/mL, MeOH);MS (ES) m/z 369.8.
Example 46:
Representative compounds of this invention were evaluated in the following
standard pharmacological test procedures which demonstrated antiinflammatory activity.
The test procedures used and the results obtained are briefly described below.
Test procedures:
T-175 flasks of 100% confluent HAECT-1 cells (immortalized human aortic
endothelial cells) were washed with 8 ml of HBSS (HEPES buffered saline solution) and
infected for four hou with 6 ml of a 1:10 dilution of Ad5-wt-hERcc virus (an adenovirus
transfection vector that mediates CMV promoter driven expression of human ERa) in
phenol red free Endothelial Cell Basal medium (Clonetics, San Diego CA, Catalog # CC-
3129) containing 0.25% bovine serum albumin (EBM-BSA). After four hou, cells were
washed with EBM-BSA and incubated overnight in the same medium. Following
overnight incubation, cells were washed with EBM-BSA and infected for 2 hou with 6
ml of a 1:10 dilution of Ad5-3x(NFicB).Luc virus (Adenovirus luciferase expression
vector driven by 3 repeats of the MHC NFicb site 5' to the thymidine kinase promoter) in
EBM-BSA. After two hou, cells were washed and incubated at 34°C for 1 hour. Cells
were then washed, trypsinized, counted and resuspended in 95%FBS / 5%
dimethylsulfoxide at a concentration of 4x106 cells/ml, frozen as 1 or 5 ml aliquots in
cryo-vials and stored at -150°C. Control (no ER infection) cells were processed as above
without Ad5-wt-hERcc virus infection.
IL-6 and Creatine Kinase Assays
ERa infected HAECT-1 cells or control cells were thawed, diluted 42x in warm
EBM-BSA, plated into 96-well plates at 0.1 ml/well and incubated for 4h at 34°C. Test
compounds were added to the cells as 2x stocks in EBM-BSA containing 2 ng/ml IL-lp
Ad5-IL-6(1250bp).Luc virus and plates were returned to the incubator (34°C). After 15-
20h, cells are lysed with 50 ul of Promega Cell Culture Lysis reagent for ~5 min at
room temp on shaker. After lysing, 15ul of lysate is transferred to luminometer plates for
luciferase determination. Luciferase activity is evaluated using a Perkin Elmer Victor2
1420 multilabel counter. Creatine kinase was determined from the rate of increase in A340
following addition of 100 uL of CK assay reagent (Sigma, cat. No 47-10) to the
remainder of the cell lysate.
Data Analyses
For ICso and ECso calculations, mean IL-6, luciferase or CK values veus logic
of the compound concentration were fitted to a four parameter logistic equation. The
ICso/ ECso value, 'Hill slope1, upper and lower limits of the curve were iteratively
estimated.
Mice
Ovariectomized C57BL/6 mice (16-20g) (laconic) were separated into groups of
8. After 5-7 days of recuperation, the mice were fed a chow diet or an atherogenic diet
(15.75% fat, 1.25% cholesterol and 0.5% sodium cholate) (Purina diet #21539). EE or
test compound was administered once daily by gavage in a methylcellulose/tween vehicle
(0.1 ml per mouse) for 5 weeks. At the end of the experimental period, the liver was
collected and uterine wet weight was recorded.
RNA Analysis
Liver total RNA was prepared by using Trizol reagent (BRL). Estrogen and
compound regulation of NF-KB target genes were verified by real time RT-PCR using an
ABI PRISM 7700 Sequence Detection System according to the manufacturer's protocol
(Applied Biosystems). The data was analyzed using the Sequence Detector vl.7 software
(Applied Biosystems) and normalized to GAPDH using the Applied Biosystems primer
The following table summarizes the results obtained in the standard
pharmacological test procedures described above:
If not otherwise indicated, the RI-RH substituent represents hydrogen.
E2 inhibits NF-KB and IL-6 expression in Ad5-wt-ER infected HAECT-1 cells
with an ICso value around 1 nM and induces expression of creatine kinase in the same
cells with similar potency (5.8 nM). In contrast, preferred compounds of the present
invention potently and efficaciously inhibit NF-KB and IL-6 expression in Ad5-wt-ER
infected HAECT-1 cells but do not induce CK expression in an ER-dependent manner.
The ability of preferred compounds of the present invention to inhibit NF-KB and IL-6
expression without inducing CK activity demonstrates anti-inflammatory activity in the
absence of classic estrogenic activity.
Based on the results obtained in the standard pharmacological test procedures, the
compounds of this invention are selective anti-inflammatory compounds useful for the
treatment and prevention of chronic inflammatory diseases without stimulating uterine
and breast cell proliferation as found with classic estrogens.
Example 47: The processes detailed below are illustrative of processes for evaluating the
compounds of the present invention.
Evaluation of test compound using an ERE-reporter test procedure in MCF-7 breast
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 °C (0.1 M) or -20 °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 °C inside a 5% CO2/95%
humidified air incubator. One day prior to treatment, the cells are plated with growth
medium at 25,000 cells/well into 96 well plates and incubated at 37 °C overnight.
The cells are infected for 2 hr at 37 °C with 50 D I/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 Dl of experimental medium. Finally, the cells are treated for
24 hr at 37 °C in replicates of 8 wells/treatment with 150 D I/well of vehicle (< 0.1% v/v
DMSO) or compound that is diluted > 1000-fold into experimental medium.
Initial screening of test compounds is done at a single dose of 1 DM that is tested
alone (estrogen receptor agonist mode) or in combination with 0.1 nM 17D-estradiol
(EC80; estrogen receptor antagonist mode). Each 96 well plate also includes a vehicle
control group (0.1% v/v DMSO) and an estrogen receptor agonist control group (either
0.1 or 1 nM 17D-estradiol). Dose-response experiments are performed in either the
estrogen receptor agonist and/or estrogen receptor antagonist modes on active compounds
in log increases from 10-14 to 10-5 M. From these dose-response curves, EC50 and IC50
values, respectively, are generated. The final well in each treatment group contains 5 Dl
of 3 x 10-5 M ICI-182,780 (10-6 M final concentration) as an estrogen receptor
antagonist control.
After treatment, the cells are lysed on a shaker for 15 min with 25 D I/well of IX
cell culture lysis reagent (Promega Corporation). The cell lysates (20 Dl) 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 D I/well of luciferase substrate (Promega
Corporation). Prior to the injection of substrate, a 1 second background measurement is
78
and water. On day 22, 23, 24 and 25 the mice are dosed subcutaneously with compound
or vehicle (corn oil). There are six mice/group and they are euthanized approximately 6
hou after the last injection by CO2 asphyxiation and pneumothorax. Uteri are removed
and weighed after trimming associated fat and expressing any internal fluid.
Evaluation of osteoporosis and lipid modulation (cardioprotection)
Female Sprague-Dawley rats, ovariectomized or sham operated, are obtained 1
day after surgery from laconic Farms (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 arrival
and rats are 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.
All test compounds are prepared in a vehicle of 50% DMSO (JT Baker,
Phillipsburg, NJ) / Ix Dulbecco's phosphate saline (GibcoBRL, Grand Island, NY) at
defined concentrations so that the treatment volume is 0.1 mL/100 g body weight.
.17Destradiol is dissolved in corn oil (20 |ag/mL) and delivered subcutaneously, 0.1
mL/rat. All dosages are adjusted at three week intervals according to group mean body
weight measurements, and given subcutaneously.
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.
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° angle and the knee joint at
180°. 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 is 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
80
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.
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 mathematically removed using an iterative algorithm. The density of the remaining
bone (total density) is reported in mg/cm3. The outer 55% of the bone is mathematically
peeled away in a concentric spiral. The density of the remaining bone (Trabecular
density) is reported in mg/cm3.
One week after BMD evaluation the rats are euthanized by CO2 asphyxiation and
pneumothorax, and blood is collected for cholesterol determination. The uteri are also
removed and the weighed after trimming associated fat and expressing any luminal fluid.
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.
Evaluation of antioxidant activity
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 oC for 15-20 minutes,
after which the collagenase solution is collected and centrifuged for 5 minutes at 2000 x
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
Dg/ml) and gentamycin (75 Dg/ml), seeded in 100 mm petri dish and incubated at 37 oC
in 5%CO2. After 20 minutes, the cells are rinsed with PBS and fresh medium added, this
was repeated again at 24 hou. 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 Dg/mL LDL is allowed to proceed
81
in the presence of the compound to be evaluated (5 DM) for 4 hou at 37 °C. Results are
expressed as the percent inhibition of the oxidative process as measured by the TEA
(thiobarbituric acid reactive substances) method for analysis of free aldehydes (Yagi,
Biochemical Medicine 15: 212-6 (1976)).
Progesterone receptor mRNA regulation standard pharmacological test procedure
This test procedure can be used to evaluate the estrogenic or antiestrogenic
activity of compounds from this invention (Shughrue, et al., Endocrinology 138: 5476-
5484 (1997)).
Rat Hot Flush Test Procedure
The effect of test compounds on hot flushes can be evaluated in a standard
pharmacological test procedure which measures the ability of a test compound to blunt
the increase in tail skin temperature which occu as morphine-addicted rats are acutely
withdrawn from the drug using naloxone (Merchenthaler, et al., Maturitas 30: 307-16
(1998)). It can also be used to detect estrogen receptor antagonist activity by co-dosing
test compound with the reference estrogen.
Evaluation of vasomotor function in isolated rat aortic rings
Sprague-Dawley rats (240-260 grams) are divided into 4 groups:
1. Normal non-ovariectomized (intact)
2. Ovariectomized (ovex) vehicle treated
3. Ovariectomized 17Destradiol treated (1 mg/kg/day)
4. Ovariectomized animals treated with test compound (various doses).
Animals are Ovariectomized approximately 3 weeks prior to treatment. Each
animal receives either 17-estradiol sulfate (1 mg/kg/day) 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.
Animals are euthanized by CO2 inhalation and exsanguination. Thoracic aortae
are rapidly removed and placed in 37 oC physiological solution with the following
composition (mM): NaCl (54.7), KC1 (5.0), NaHC03 (25.0), MgC12 2H2O (2.5), Dglucose
(11.8) and CaC12 (0.2) gassed with CO2-O2, 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.
82
Rings are suspended in a 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.
After equilibration, the rings are exposed to increasing concentrations of
phenylephrine (10-8 to 10-4 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.
Evaluation of cardioprotective activity
Apolipoprotein E-deficient C57/B1J (apo E KO) mice are obtained from laconic
Farms. All animal procedures are performed under strict compliance to IACUC
guidelines. Ovariectomized female apo E KO mice, 4-7 weeks of age, are housed in shoebox
cages and were allowed free access to food and water. The animals are randomized
by weight into groups (n=12-15 mice per group). The animals are dosed with test
compounds or estrogen (17|3-estradiol sulfate at 1 mg/kg/day) in the diet using a Precisedosing
Protocol, where the amount of diet consumed is measured weekly, and the dose
adjusted accordingly, based on animal weight. The diet used is a Western-style diet
(57U5) that is prepared by Purina and contains 0.50% cholesterol, 20% lard and 25
IU/KG Vitamin E. The animals are dosed/fed using this paradigm for a period of 12
weeks. Control animals are fed the Western-style diet and receive no compound. At the
end of the study period, the animals are euthanized and plasma samples obtained. The
hearts are perfused in situ, fit with saline and then with neutral buffered 10% formalin
solution.
For the determination of plasma lipids and lipoproteins, total cholesterol and
triglycerides are determined using enzymatic methods with commercially available kits
from Boehringer Mannheim and Wako Biochemicals, respectively and analyzed using
the Boehringer Mannheim Hitachii 911 Analyzer. Separation and quantification of
plasma lipoproteins were performed using FPLC size fractionation. Briefly, 50-100 mL
of serum is filtered and injected into Superose 12 and Superose 6 columns connected in
series and eluted at a constant flow rate with 1 mM sodium EDTA and 0.15 M NaCl.
Areas of each curve representing VLDL, LDL and HDL are integrated using Wate
83
Millennium™ software, and each lipoprotein fraction is quantified by multiplying the
Total Cholesterol value by the relative percent area of each respective chromatogram
3eak.
For the quantification of aortic atherosclerosis, the aortas are carefully isolated
and placed in formalin fixative for 48-72 hou before handling. Atherosclerotic lesions
are identified using Oil Red O staining. The vessels are briefly destained, and then
imaged using a Nikon SMU800 microscope fitted with a Sony 3CCD video camera
system in concert with IMAQ Configuration Utility (National Instrument) as the image
capturing software. The lesions are quantified en face along the aortic arch using a
custom threshold utility software package (Coleman Technologies). Automated lesion
assessment is performed on the vessels using the threshold function of the program,
specifically on the region contained within the aortic arch from the proximal edge of the
brachio-cephalic trunk to the distal edge of the left subclavian artery. Aortic
atherosclerosis data are expressed as percent lesion involvement strictly within this
defined luminal area.
Evaluation of cognition enhancement
Ovariectomized rats (n=50) are habituated to an 8-arm radial arm maze for 10-
min periods on each of 5 consecutive days. Animals are water-deprived prior to
habituation and testing. A 100 uL aliquot of water placed at the ends of each arm serves
as reinforcement. Acquisition of a win-shift task in the radial arm maze is accomplished
by allowing the animal to have access to one baited arm. After drinking, the animal exits
the arm and re-ente the central compartment, where it now has access to the previously
visited arm or to a novel arm. A correct response is recorded when the animal chooses to
enter a novel arm. Each animal is given 5 trials per day for 3 days. After the last
acquisition trial, the animals are assigned to one of the following 4 groups:
1. Negative controls: injected with 10% DMSO/ sesame oil vehicle once daily for
6 days (1 mL/kg, SC)
2. Positive controls: injected with 17D-estradiol benzoate for 2 days and tested 4
days after the second injection (17D-estradiol benzoate at 10 ng/0.1 mL per rat)
3. Estradiol: 17D-estradiol will be injected daily for 6 days (20 ng/kg, SC)
4. Test compound: injected daily for 6 days (doses vary).
84
All injections will begin after testing on the last day of acquisition. The last
injection for groups 1, 3, and 4 will take place 2 hou before testing for working
memory.
The test for working memory is a delayed non-matching-to-sample task (DNMS)
utilizing delays of 15, 30, or 60 seconds. This task is a variation of the acquisition task in
which the rat is placed in the central arena and allowed to enter one arm as before. A
second arm is opened once the rat travees halfway down the fit arm, and again the rat
is required to choose this arm. When it has traveled halfway down this second arm, both
doo are closed and the delay is instituted. Once the delay has expired, both of the
original two doo, and a third novel door, are opened simultaneously. A correct
response is recorded when the animal travels halfway down the third, novel arm. An
incorrect response is recorded when the animal travels halfway down either the fit or
second arms. Each animal will receive 5 trials at each of the three delay intervals for a
total of 15 trials per subject.
Evaluation of effect on pleurisy
The ability to reduce the symptoms of experimentally-induced pleurisy in rats can
be evaluated according to the procedure of Cuzzocrea (Endocrinology 141: 1455-63
(2000)).
Evaluation of protection against glutamate-induced cytotoxicity (neuroprotection)
The neuroprotective activity of compounds of this invention can be evaluated in
an in vitro standard pharmacological test procedure using glutamate challenge
(Zaulyanov, et al., Cellular & Molecular Neurobiology 19: 705-18 (1999); Prokai, et al.,
Journal of Medicinal Chemistry 44: 110-4 (2001)).
Evaluation in the Mammary End Bud Test Procedure
Estrogens are required for full ductal elongation and branching of the mammary
ducts, and the subsequent development of lobulo-alveolar end buds under the influence of
progesterone. In this test procedure, the mammotrophic activity of selected compounds
of the invention can be evaluated according to the following standard pharmacological
test procedure. Twenty-eight day old Sprague-Dawley rats (Taconic Farms,
Germantown, NY) are ovariectomized and rested for nine days. Animals are housed
under a 12-hour light/dark cycle, fed a casein-based Purina Laboratory Rodent Diet 5K96
(Purina, Richmond, IN) and allowed free access to water. Rats were then dosed
subcutaneously for six days with vehicle (50% DMSO (JT Baker, Phillipsburg, NJ) / 50%
Ix Dulbecco's Phosphate buffered saline (GibcoBRL, Grand Island, NY), 17p-estradiol
(0.1 mg/kg) or test compound (20 mg/kg). For the final three days, rats are also dosed
subcutaneously with progesterone (30 mg/kg). On the seventh day, rats are euthanised
and a mammary fat pad excised. This fat pad is analyzed for casein kinase II mRNA as a
marker of end bud proliferation. Casein kinase II mRNA is analyzed by real-time RTPCR.
Briefly, RNA is isolated following Trizol (GibcoBRL, Grand Island, NY)
according to the manufacture's directions, Samples are treated with DNAse I using
DNA-free kit (Ambion), and casein kinase II mRNA levels are measured by real-time
RT-PCR using the Taqman Gold procedure (PE Applied Biosystems). A total of 50 ng of
RNA is analyzed in triplicate using casein kinase II specific primer pair (5' primer,
CACACGGATGGCGCATACT (SEQ ID NO. 1); 3' primer,
CTCGGGATGCACCATGAAG (SEQ ID NO. 2)) and customized probe (TAMRACGGCACTGGTTTCCCTCACATGCT-
FAM (SEQ ID NO. 3)). Casein kinase II
mRNA levels are normalized to 18s ribosomal RNA contained within each sample
reaction using prime and probe supplied by PE Applied Biosystems.
Evaluation in the HLA Rat Standard Pharmacological Test Procedure for inflammatory
bowel disease
Representative compounds can be evaluated in the HLA rat standard
pharmacological test procedure which emulates inflammatory bowel disease in humans.
The following briefly describes the procedure used and results obtained. Male HLA-B27
rats are obtained from Taconic and provided unrestricted access to food (PMI Lab diet
5001) and water. Rats are dosed subcutaneously once per day with either vehicle (50%
DMSO / 50% Ix Dulbecco's Phosphate Buffered Saline) or test compound (0.1 to 10
mg/kg) for at least one week. 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 study,
serum is collected and stored at -70 °C. A section of colon is prepared for histological
analysis and an additional segment is analyzed for myeloperoxidase activity.
For histological analysis, colonic tissue is immeed in 10% neutral buffered
formalin. Each specimen of colon is separated into four samples for evaluation. The
formalin-fixed tissues are processed in a Tissue Tek vacuum infiltration processor (Miles,
Inc; West Haven, Connecticut) for paraffin embedding. The samples are sectioned at 5
um 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. Sections of colonic tissue are evaluated for
several disease indicato and given relative scores.
Evaluation in three models of arthritis
Lewis rat assay of adjuvant-induced arthritis. Sixty, female, 12 weeks old, Lewis
rats are housed according to standard facility operating procedures. They receive a
standard regimen of food and water ad libitum. Each animal is identified by a cage
card indicating the project group and animal number. Each rat number is marked by
indelible ink marker on the tail. At least 10-21 days before study they are anesthetized
and ovariectomized by standard aseptic surgical techniques.
Freund's Adjuvant-Complete (Sigma Immuno Chemicals, St. Louis, MO) is used
to induce arthritis, each mL containing 1 mg Mycobacterium tuberculosis heat killed and
dried, 0.85 mL mineral oil and 0.15 mL mannide monooleate Lot No. 084H8800.
The following are examples of two test procedures. Inhibition test procedure:
Thirty rats are injected intradermally with 0.1 mL of Freund's Adjuvant-Complete at the
base of the tail. The animals are randomized to four groups, each group containing six
rats. Each day, the groups receive vehicle (50% DMSO (JT Baker, Phillipsburg, NJ) / Ix
Dulbecco's phosphate saline (GibcoBRL, Grand Island, NY)) or test compound (0.1-10
mg/kg, administered subcutaneously). All rats begin treatment on Day 1.
Treatment test procedure: Thirty rats are injected intradermally with 0.1 mL of
Freund's Adjuvant-Complete at the base of the tail. The animals are randomized to four
groups, each group containing six rats. Each day, the groups receive vehicle (50%
DMSO (JT Baker, Phillipsburg, NJ) / Ix Dulbecco's phosphate saline (GibcoBRL, Grand
Island, NY)) or test compound (0.1-10 mg/kg, administered subcutaneously). All rats
begin treatment on Day 8 after adjuvant injection.
Statistical analysis is performed using Abacus Concepts Super ANOVA. (Abacus
Concepts, Inc., Berkeley, CA). All of the paramete of interest are subjected to Analysis
of Variance with Duncan's new multiple range post hoc testing between groups. Data
are expressed throughout as mean ± standard deviation (SD), and differences are deemed
significant if p<0.05.
The degree of arthritis severity is monitored daily in terms of the following
disease indices: Hindpaw erythema, hindpaw swelling, tenderness of the joints, and
movements and posture. An integer scale of 0 to 3 is used to quantify the level of
erythema (0= normal paw, 1= mild erythema, 2= moderate erythema, 3= severe
erythema) and swelling (0=normal paw, l=mild swelling, 2= moderate swelling, 3=
severe swelling of the hind paw). The maximal score per day is 12.
At the end of the study the rats are euthanized with CO2, hindlimbs removed at
necropsy and fixed in 10% buffered formalin, and the taal joints decalcified and
embedded in paraffin. Histologic sections are stained with Hematoxylin and Eosin or
Saffranin O - Fast Green stain.
Slides are coded so that the examiner is blinded to the treatment groups. Synovial
tissue from taal joints is evaluated based on synovial hyperplasia, inflammatory cell
infiltration, and pannus formation (Poole and Coombs, International Archives of Allergy
& Applied Immunology 54: 97-113 (1977)) as outlined below.
Category
1 . Synovial lining cells
a. No change
b. Cells enlarged, slightly thickened
c. Cells enlarged, increase in numbe, moderately thickened. No villus
present
d. Cells enlarged, thickened. Villlus present
2. Fibroplasia
a. No change
b. Fibroplasia present under lining cells
c. Small areas of areolar tissue replaced by fibrous tissue
d. Replacement of areolar tissue by fibrous tissue
3. Inflammatory cells
a. Occasionally seen, scattered throughout selection
b. Cells present in small numbe in or just under lining cell layer
and/or around blood vessels.
c. Small focal collection of cells may be present
d. Large numbe of cells present in capsule and in or under lining cell
laye. Large foci often seen.
In addition, articular cartilage and bone is evaluated using Mankin's histological
grading system (Mankin, et al., Journal of Bone & Joint Surgery - American Volume 53:
523-37 (1971)) as shown below.
Category
1. Structure
a. Normal
b. Surface irregularity
c. Pannus and surface irregularity
d. Clefts to transitional zone
e. Clefts to radial zone
f. Clefts to calcified zone
g. Complete disorganization
2. Cells
a. Normal
b. Diffuse hypercellularity
c. Cloning
d. Hypocellularity
3. Safranin-O staining
a. Normal
b. Slight reduction
c. Modest reduction
d. Severe reduction
e. No dye noted
4. Tidemark integrity
a. Intact
b. Crossed by blood vessels
Evaluation in the HLA-B27 Rat model of arthritis.
Representative compounds are evaluated in the HLA-B27 rat standard
pharmacological test procedure which emulates arthritis in humans. The following
briefly describes the procedure used. Male HLA-B27 rats are obtained from Taconic and
provided unrestricted access to a food (PMI Lab diet 5001) and water. Rats are dosed
subcutaneously once per day with either vehicle (50% DMSO / 50% Ix Dulbecco's
Phosphate Buffered Saline) or test compound (0.1 to 10 mg/kg) for at least one week.
Joint scores and histology are evaluated as described above for the Lewis rat model of
adjuvant-induced arthritis.
Evaluation in the collagen induced arthritis models.
Compounds are evaluated in BALB/c mice, 6-8 weeks of age, in which arthritis is
induced by monoclonal antibodies raised against type II collagen, plus lipopolysaccharide
(LPS). The animals were administered intravenously with a combination of 4 different
mAbs totaling 4 mg/mouse on day 0, and followed by intravenous 25 Dg of LPS 72
hou later (day 3). From day 3, one hour after LPS application, tested compounds are
give orally once daily for 15 days. For each animal, increase in volume of both hind
paws is measured using a plethysmometer with water cell (12 mm diameter) on days 0, 5,
7, 10, 14 and 17. Percent inhibition of increase in volume is calculated.
Evaluation in vivo models of carcinogeneisis
The ability of compounds of this invention to treat and inhibit various
malignancies or hyperprolific disorde can be evaluated in standard pharmacological test
procedures that are readily available in the literature, and include the following two
procedures.
Breast cancer. Athymic nu/nu (nude) mice are obtained ovariectomized from
Charles River Laboratories (Wilmington, MA). One day prior to tumor cell injection,
animals are implanted with time-release pellets containing 0.36-1.7 mg 17p-estradiol (60
or 90 day release, Innovative Research of America, Sarasota, FL) or a placebo. The
pellet is introduced subcutaneously into the intrascapular region using a 10-gauge
precision trochar. Subsequently, mice are injected subcutaneously into the breast tissue
with either 1x107 MCF-7 cells or 1x107 BG-1 cells. The cells are mixed with an equal
volume of matr1gEl, a basement membrane matrix preparation to enhance tumor
establishment. Test compounds can be evaluated either by dosing one day after tumor
cell implantation (inhibition regimen) or after tumo have reached a certain size
(treatment regimen). Compounds are administered either intraperitoneally or orally in a
vehicle of 1% tween-80 in saline each day. Tumor size is evaluated every three or seven
days.
[0002] Colon cancer. The ability to treat or inhibit colon cancer can be evaluated
in the test procedure of Smirnoff (Oncology Research 11: 255-64 (1999)).
Evaluation of neuroprotection in two in vivo test procedures
Transient global ischemia in the Mongolian gerbil. The effect of test compounds
on preventing or treating brain injury in response to oxygen deprivation/reperfusion can
be measured using the following test procedure.
Female Mongolian gerbils (60-80 g; Charles River Laboratories, Kingston, NY)
are housed in the Wyeth-Ayet animal care facility (AAALAC certified) with a 12-hour
light, 12-hour dark photoperiod and free access to tap water and a low-estrogen casein
diet (Purina; Richmond, IN). After acclimation (3-5 days), gerbils are anesthetized with
isoflurane (2-3% mixture with 02), ovariectomized (Day 0). Beginning the following
morning (Day 1), gerbils are treated subcutaneously each day with either vehicle (10%
ETOH/corn oil), 17p-estradiol (1 mg/kg) or an experimental compound (0.1-20 mg/kg).
On Day 6, gerbils (n=4-5/group) are anesthetized with isoflurane, the common carotid
arteries visualized via a mid-line neck incision and both arteries simultaneously occluded
for 5 minutes with non-traumatic micro aneurysm clips. After occlusion, the clips are
removed to allow cerebral reperfusion and the neck incision closed with wound clips. All
animals are fasted overnight prior to the global ischemia surgery, a step that facilitates
consistent ischemic injury. On Day 12, gerbils are exposed to a lethal dose of CO2, and
the brains frozen on dry ice and stored at -80 °C.
The degree of neuronal protection is evaluated by in situ hybridization analysis of
neurogranin mRNA. Briefly, 20 um coronal cryostat sections are collected on gelatincoated
slides, dried and stored at -80 °C. At the time of processing, the desiccated slide
boxes are wanned to room temperature, the slides postfixed in 4% paraformaldehyde,
treated with acetic anhydride and then delipidated and dehydrated with chloroform and
ethanol. Processed section-mounted slides are then hybridized with 200 ul (6x106 DPMI
slide) of an antisense or sense (control) riboprobe for Neurogranin (35S-UTP-labeled
NG-241; bases 99-340). in a 50% formamide hybridization mix and incubated overnight
at 55 °C in a humidified slide chamber without covelipping. The following morning,
the slides are collected in racks, immeed in 2xSSC (0.3 M NaCl, 0.03 M sodium citrate;
pH 7.0) / 10 mM DTT, treated with RNase A (20 ug/ml) and washed (2 x 30 min) at 67
°C in 0.1 x SSC to remove nonspecific label. After dehydration, the slides are opposed to
BioMax (BMR-1; Kodak) X-ray film overnight.
The level of neurogranin hybridization signal is used to quantitatively assess the
degree of neuronal loss in the CA1 region after injury and to evaluate the efficacy of 170-
estradiol and experimental compounds. Neurogranin mRNA is selected for these studies
because it is highly expressed in the hippocampal neurons including CA1, but absent in
glia and other cell types present in this brain region. Therefore, measurement of the
amount of neurogranin mRNA present represents surviving neurons. Relative optical
density measurements of neurogranin hybridization signal are obtained from film
autoradiograms with a computer based image analysis system (C-Imaging Inc.,
Pittsburgh, PA). The results from 6 sections (40 um apart) per animal are averaged and
statistically evaluated. Numerical values are reported as the mean + SEM. One-way
analysis of variance is used to test for differences in the level of neurogranin mRNA and
all statements of non-difference in the results section imply that p>0.05.
Middle cerebral artery occlusion in mice. Neuroprotection can be evaluated
according to the test procedures described by Dubai (see, Dubai, et al., Proceedings of the
National Academy of Sciences of the United States of America 98: 1952-1957 (2001),
Dubai, et al., Journal of Neuroscience 19: 6385-6393 (1999)).
Ovulation inhibition standard pharmacological test procedure
The test procedure is used to determine whether test compounds can inhibit or
change the timing of ovulation. It can also be used to determine the number of oocytes
ovulated [Lundeen, et al., J Steroid Biochem Mol Biol 78: 137-143 (2001)].
Transplantation Rejection
To test the ability of the test compounds to prevent transplant rejection.
Compounds can be tested in animal models of heart transplantation (Stetson et al.
Circulation 104:676-682 (2001) or transplant atherosclerosis (Deitrich et al. Arterioscler.
Thromb. Vase Biol. 20:343-352 (2000), Lou et al., Circulation 94:3355-3361 (1996).
Prevention of Restenosis
The test procedure is used to determine whether test compounds can inhibit
vascular smooth muscle cell proliferation after carotid artery injury similar to what occu
after balloon angioplasty. The test compounds can be tested in animal models previously
described (Karas et al. Circ Res. 89:534-539 (2001), Cerek et al. Atherosclerosis 131:59-
66(1997).
Treatment of Myocardial Infarction
Test compounds can be tested in animal models of ischemia/reperfusion to
determine whether they would inhibit cell death occurring during a myocardial infarction.
The compounds can be tested in models described previously (Delyani et al. J Mol & Cell
Cardiology 28:1001-1008 (1996), Izumi et al. J Clin Invest. 108:203-213 (2001) &
Chandrasekar et al. Circulation 103:2296-2302 (2001)).
Treatment for Myocarditis and Congestive Heart Failure
Test compounds can be tested in models of heart failure to determine whether
compounds could be an effective therapy and improve cardiac function. Compounds can
be tested in animals as described previously (Yokoseki et al. Circ Res. 89:1-9 (2001),
Wallen et al. Hypertension 36:774-779 (2000) & Toshiaki et al. Circulation 104:1094-
1103(2001)).
Treatment for Diabetes
Test compounds can be tested in models of diabetes to determine their effect on
reveal of obesity and diet-induced insulin resistance. Compounds can be tested in
animal models as previously described (Yuan et al. Science 293:1673-1677 (2001).
Treatment for Asthma
Pulmonary Inflammation Model Mice are sensitized with OVA emulsified in
alum on days 0 and 14 (ip injection). On days 28 and 29, mice are challenged with an
aerosol of OVA for 20 min (l%-5% OVA) and then on Day 30 the animals are sacrificed
and harvest BAL and/or lung tissue for analysis of pulmonary inflammation.
Airway Hyperresponsiveness. This model is similar to that described above
however animals are challenged on 3 consecutive days with an aerosol of OVA and
airway hyperresponsiveness is measured 48 h after the last challenge. BAL can also be
taken at this stage if required.
To look more directly at the effects of mast cells in conjunction with ER, a
passive cutaneous anaphylaxis model in which 1gE is injected into the ear and then 24
hou later DNP-HSA iv is injected DNP-HSA iv can be used. Ear thickness and an early
and late phase reaction are measured. Tissues are fixed in K2. embedded in Epoxy resin
and cut into luum sections. These can be stained for mast cells and the degree of mast
cell degranulation can be quantified.
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 peonal 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.
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 estrogen receptor
agonist mode, or the positive estrogen receptor agonist control results (0.1 nM 170-
estradiol) in the estrogen receptor 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
17p-estradiol control [i.e., ((compound - vehicle control)/(17p-estradiol control - vehicle
control)) x 100]. The JMP software is also used to determine the EC50 and/or IC50
values from the non-linear dose-response curves.
Evaluation of uterotrophic activity
Uterotrophic activity of a test compound can be measured according to the
following standard pharmacological test procedures.
Procedure 1: Sexually immature (18 days of age) Sprague-Dawley rats are
obtained from Taconic and provided unrestricted access to a casein-based diet (Purina
Mills 5K96C) and water. On day 19, 20 and 21 the rats are dosed subcutaneously with
17a-ethinyl-17p-estradiol (0.06 g/rat/day), test compound or vehicle (50% DMSO/50%
Dulbecco's PBS). To assess estrogen receptor antagonist, compounds are coadministered
with 17cc-ethinyl-17p-estradiol (0.06 ug/rat/day). There are six rats/group and they are
euthanized approximately 24 hou after the last injection by CO2 asphyxiation and
pneumothorax. Uteri are removed and weighed after trimming associated fat and
expressing any internal fluid. A tissue sample can also be snap frozen for analysis of
gene expression (e.g. complement factor 3 mRNA).
Procedure 2: Sexually immature (18 days of age) 129 SvE mice are obtained from
Taconic and provided unrestricted access to a casein-based diet (Purina Mills 5K96C)

WE CLAIM:
1. A compound of Formula 1:
or a pharmaceutically acceptable salt or ester form thereof
(Figure Remove) wherein
R1, R2, RS, and R4, are, independently, hydrogen, optionally substituted lower
alkyl, halogen, or optionally substituted aryl;
R7, R8, R9, and RIO, are, independently, hydrogen, optionally substituted lower
alkyl or halogen;
R1, R12, RH, and RIS are, independently, hydrogen, hydroxy, optionally
substituted lower alkyl, optionally substituted alkoxy, or halogen;
one of RS and R is independently, hydrogen and the other is optionally
substituted lower alkyl;
R13 is hydrogen, -(C=O)Ri6, -S(O)2Ri7, -S(O)2N(Ri8)(Ri9), or D-glucuronidate;
Ri6 is optionally substituted alkyl, optionally substituted arylalkyl or optionally
substituted aryl;
is optionally substituted alkyl, optionally substituted aryl, optionally
substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted alkenyl,
optionally substituted cycloalkenyl, or optionally substituted alkynyl;
Rig and Ri9 are, independently, hydrogen, optionally substituted alkyl, optionally
substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl,
monofluoroalkyl, perfluoroalkyl, optionally substituted aryl, optionally substituted
arylalkyl, optionally substituted cycloalkenyl, optionally substituted heteroaryl,
optionally substituted heteroarylalkyl, optionally substituted hydroxy-(C2-C6)alkyl,
optionally substituted alkoxyalkyl, optionally substituted alkylthioalkyl, carbonyl,
optionally substituted acyl, optionally substituted alkoxycarbonyl, -C(0)NH2, optionally
substituted alkylammocarbonyl, optionally substituted dialkylaminocarbonyl, optionally
substituted alkylaminoalkyl, or optionally substituted dialkylaminoalkyl;
or Rig and R are taken together with the nitrogen atom to which they are
attached to form a saturated, unsaturated or partially saturated C4-C6 carbon ring.
2. A compound of claim 1 having Formula 2 or Formula 3:
Formula 2 Formula 3
or a pharmaceutically acceptable salt or ester form thereof.
3. A compound of claim 1 having Formula 4:
or a pharmaceutically acceptable salt or ester form thereof.
4. A compound of claim 3 having Formula 5 or 6:
or a pharmaceutically acceptable salt or ester form thereof.
5. A compound of Claim 1 having Formula 7:
or a pharmaceutically acceptable salt or ester form thereof.
6. A compound of claim 5 having Formula 8 or 9:
or a pharmaceutically acceptable salt or ester form thereof.
8. A compound of claim 7 having Formula 11 or 12:
Formula 11 Formula 12
or a pharmaceutically acceptable salt or ester form thereof.
9. A compound as claimed in any one of claims 1 to 8 wherein at least one of Rj,
A compound of claim 9 wherein RI, R2, Ra, RI, R?, R«, Rg, and RIO are hydrogen
or halogen.
11. A compound as claimed in claim 9 or claim 10 wherein at least one of RI or Rg
is halogen.
12. A compound of claim 1 0 wherein RS and Rg are halogen.
13. A compound of claim 10 wherein RS or Rg is chlorine or fluorine or RS and Rg
are chlorine or flourine.
14. A compound of any one of claims 1 to 13 wherein Ri2, RH and RIS are
hydrogen, halogen, hydroxy, or alkoxy
15. A compound of claim 14 wherein R, RM and RIS are hydrogen, chlorine,
bromine, fluorine, hydroxy, or methoxy.
16. A compound of any one of claims 1 to 8 wherein at least one of RI, RI, RS, R4,
R7, Rg, Rg, and RIO, is halogen, Rn, RH and RIS are hydrogen, chlorine, bromine,
fluorine, hydroxy, or methoxy and RIS is hydrogen.
17. A compound of claim 1 that is 4-{[(6)-8-fluoro-6-methylphenanthridin-5(6/)-
yl]carbonyl}phenol; 3-{[(65)-8-fluoro-6-methylphenanthridin-5(6//)-yl]carbonyl}phenol;
4-{[(65)-8-fluoro-6-methylphenanthridin-5(6//)-yl]carbonyl}phenol; 3-{[(6fl)-8-fluoro-
6-methylphenanthridin-5(6//)-yl]carbonyl}phenol; 3-fluoro-4-{[(6?)-8-fluoro-6-
methylphenanthridin-5(6//)-yl]carbonyl}phenol; 3-fluoro-4-{[(65')-8-fluoro-6-
methylphenanthridin-5(6//)-yl]carbonyl}phenol; or a pharmaceutically acceptable salt,
ester, or solvate form thereof.
18. A compound as claimed in claim 1 that is 4-fluoro-3-{[(6/?)-8-fluoro-6-
methylphenanthridin-5(6//)-yl]carbonyl}phenol; 4-fluoro-3- {[(6S)-8-fluoro-6-
methylphenanthridin-5(6//)-yl]carbonyl}phenol; 2-fluoro-4- {[(6Jl?)-8-fluoro-6-
methylphenanthridin-5(6//)-yl]carbonyl}phenol;2-fluoro-4-{[(6S)-8-fluoro-6-
methylphenanthridin-5(6/T)-yl]carbonyl}phenol;2-chloro-5-{[(6J?)-8-fluoro-6-
methylphenanthridin-5(6/:0-yl]carbonyl}phenol; or a pharmaceutically acceptable salt,
ester, or solvate form thereof.
19. A compound as claimed in claim 1 that is 2-chloro-5-{[(6iS)-8-fluoro-6-
methylphenanthridin-5(6#)-yl]carbonyl}phenol; 2-bromo-4-{[(6/?)-8-fluoro-6-
methylphenanthridin-5(6//)-yl]carbonyl}phenol; 2-bromo-4-{[(65)-8-fluoro-6-
methylphenanthridin-5(6/f)-yl]carbonyl}phenol; 4-bromo-3-{[(6J?)-8-fluoro-6-
methylphenanthridin-5(6/f)-yl]carbonyl}phenol; 4-bromo-3-{[(6S)-8-fluoro-6-
methylphenanthridin-5(6/0-yl]carbonyl}phenol; or a pharmaceutically acceptable salt,
ester, or solvate form thereof.
20. A compound as claimed in claim 1 that is 4-{[(6/?)-8-fluoro-6-
methylphenanthridin-5(6//)-yl]carbonyl}-2-methoxyphenol;4-{[(6S)-8-fluoro-6-
methylphenanthridin-5(6/0-yl]carbonyl}-2-methoxyphenol;4-{[(6S)-6-ethyl-8-
fluorophenanthridin-5(6//)-yl]carbonyl}phenol;4-{[(6)-6-ethyl-8-fluorophenanthridin-
5(6/0-yl]carbonyl}phenol;3-{[(6)-6-ethyl-8-fluorophenanthridin-5(6/f)-
yl]carbonyl}phenol; or a pharmaceutically acceptable salt, ester, or solvate form thereof.
21. A compound as claimed in claim 1 that is 4-{[(6/?)-6-ethyl-8-
fluorophenanthridin-5(6/0-yl]carbonyl}benzene-l,3-diol;4-[(6-ethyl-8-
fluorophenanthridin-5(6/f)-yl)carbonyl]-3-fluorophenol; 3-[(3-chloro-6-
methylphenanthridin-5(6//)-yl)carbonyl]phenol; 3-{[(6/?)-3-chloro-6-
methylphenanthridin-5(6/f)-yl]carbonyl}phenol; 3-{[(65)-3-chloro-6-
methylphenanthridin-5(6/f)-yl]carbonyl}phenol; or a pharmaceutically acceptable salt,
ester, or solvate form thereof.
22. A compound as claimed in claim 1 that is 4-[(3-chloro-6-methylphenanthridin-
5(6//)-yl)carbonyl]phenol;4-{[(6)-3-chloro-6-methylphenanthridin-5(6//)-
yl]carbonyl}phenol; 4-{[(65)-3-chloro-6-methylphenanthridin-5(6//)-
yl]carbonyl}phenol;
4-[3-chloro-6-methylphenanthridin-5(6/0-yl]carbonyl]benzene-l,3-diol;4-{[(6/?)-3-
chloro-6-methylphenanthridin-5(6//)-yl]carbonyl}benzene-l ,3-diol; or a
pharmaceutically acceptable salt, ester, or solvate form thereof.
23. A compound as claimed in claim 1 that is 4-{[(6S)-3-chloro-6-
methylphenanthridin-5(6ff)-yl]carbonyl}benzene-1,3-diol; 3-[(3-fluoro-6-
methylphenanthridin-5(6//)-yl)carbonyl]phenol; 3-{[(6R)-3-fiuoro-6-
methylphenanthridin-5(6H)-y\]carbonyl}phenol; 3 - {[(65)-3 -fluoro-6-
methylphenanthridin-5(6/f)-yl]carbonyl}phenol; 4-[(3-fluoro-6-methylphenanthridin-
5(6//)-yl)carbonyl]phenol; or a pharmaceutically acceptable salt, ester, or solvate form
thereof.
24. A compound as claimed in claim 1 that is 4-{[(6.K)-3-fluoro-6-
methylphenanthridin-5 (6/f)-yl] carbonyl} phenol; 4- {[(6S)-3 -fluoro-6-
methy lphenanthridin-5 (6//)-yl] carbonyl} phenol
4-[(3-fluoro-6-methylphenanthridin-5(6W)-yl)carbonyl]benzene-1,3 -diol; 4- {[(6/?)-3 -
fluoro-6-methylphenanthridin-5(6//)-yl]carbonyl}benzene-l,3-diol;4-{[(6iS)-3-fluoro-6-
methylphenanthridin-5(6/f)-yl]carbonyl}benzene-l,3-diol; or a pharmaceutically
acceptable salt, ester, or solvate form thereof.
25. A compound of claim 1 that is 4-[(3,8-difluoro-6-methylphenanthridin-5(6//)-
yl)carbonyl]phenol;4-{[(6)-3,8-Difluoro-6-methylphenanthridin-5(6//)-
yl]carbonyl}phenol; 4- {[(6S)-3,8-Difluoro-6-methylphenanthridin-5(6/f)-
yl]carbonyl}phenol; or a pharmaceutically acceptable salt, ester, or solvate form thereof.
26. A compound of claim 1 that is 4-[(3,8-difluoro-6-methylphenanthridin-5(6//)-
yl)carbonyl]phenol; 4-{[(6/?)-3,8-Difluoro-6-methylphenanthridin-5(6//)-
102
yl]carbonyl}phenol;4-{[(65)-3,8-Difluoro-6-methylphenanthridin-5(6//)-
yl]carbonyl}phenol;4-{[((5/?)-3,8-difluoro-6-methylphenanthridin-5(5//)-yl]carbonyl}-2-
fluorophenol; or a pharmaceutically acceptable salt, ester, or solvate form thereof.
27. A method of modulating cytokine expression in a subject comprising providing
to the subject a pharmaceutically effective amount of a compound as claimed in any one
of claims 1 to 26.
28. The method as claimed in claim 27 wherein said cytokine is IL-6.
29. The method as claimed in claim 27 wherein said subject has chronic inflammatory
disease.
30. The method as claimed in claim 29 wherein said chronic inflammatory
disease is atherosclerosis, myocardial infarction, congestive heart failure, inflammatory
bowel disease, or arthritis.
31. A method as claimed in treating chronic inflammatory disease comprising
providing to a subject in need thereof a pharmaceutically effective amount of a
compound as claimed in any one of claims 1 to 26.
32. A method as claimed in treating rheumatoid arthritis,
spondyloarthropathies, osteoarthritis, psoriatic arthritis, or juvenile arthritis comprising
providing to a subject in need thereof a pharmaceutically effective amount of a
compound as claimed in any one of claims 1 to 26.
33. A method as claimed in treating inflammatory bowel disease, Crohn's
disease, uleerative colitis, or indeterminate colitis comprising providing to a subject in
need thereof a pharmaceutically effective amount of a compound as claimed in any one
of claims 1 to 26.
34. A method as claimed in treating psoriasis comprising providing to a
subject in need thereof a pharmaceutically effective amount of a compound as claimed in
any one of claims 1 to 26.
35. A method as claimed in treating asthma or chronic obstructive pulmonary
disease comprising providing to a subject in need thereof a pharmaceutically effective
amount of a compound as claimed in any one of claims 1 to 26.
36. A method as claimed in treating stroke, ischemia, or reperfusion injury
comprising providing to a subject in need thereof a pharmaceutically effective amount of
a compound as claimed in any one of claims 1 to 26.
37. A method as claimed in lowering cholesterol, triglycerides, Lp(a), and
LDL levels; inhibiting or treating hypercholesteremia, hyperlipidemia, cardiovascular
disease, atherosclerosis, acute coronary syndrome, peripheral vascular disease, restenosis,
or vasospasm comprising providing to a subject in need thereof a pharmaceutically
effective amount of a compound as claimed in any one of claims 1 to 26.
38. A method as claimed in treating Alzheimer's disease, cognitive decline, or
senile dementia comprising providing to a subject in need thereof a pharmaceutically
effective amount of a compound as claimed in any one of claims 1 to 26.
39. A method as claimed in treating type II diabetes comprising providing to a
subject in need thereof a pharmaceutically effective amount of a compound as claimed in
any one of claims 1 to 26.
40. A method as claimed in treating sepsis in a mammal comprising providing
to a subject in need thereof a pharmaceutically effective amount of a compound as
claimed in any one of claims 1 to 26.
41. A kit for modulating cytokine expression in a subject, the kit comprising a
container, a pharmaceutical composition contained therein comprising a compound as
claimed in any one of claims 1 to 26.
42. A kit for treating chronic inflammatory disease in a subject, the kit
comprising a container, a pharmaceutical composition contained therein comprising a
compound as claimed in any one of claims 1 to 26.
43. A composition comprising a compound as claimed in any one of claims 1
to 26 and a pharmaceutically acceptable carrier.
44. A method comprising contacting a compound as claimed in any one of
claims 1 to 26 with a cell.
45. The method as claimed in claim 44 wherein the cell is characterized by an
overexpression of cytokine.
46. The method as claimed in claim 45 wherein said cytokine is IL-6.
47. The method as claimed in claim 44 further comprising determining the
level of expression of said cytokine.
48. The method as claimed in claim 47 wherein said determining step is made
before said contacting step.
49. The method as claimed in claim 47 wherein said determining step is made
after said contacting step.
50. A compound as claimed in any one of claims 1 to 26 for use as a
medicament.
105
51. Use of a compound as claimed in any one of claims 1 to 26 in the
preparation of a medicament for the modulation of cytokine expression in a subject.
52. Use as claimed in claim 51 wherein said cytokine is IL-6.
53. Use as claimed in claim 51 for the treatment of chronic inflammatory
disease.
54. Use of a compound as claimed in any one of claims 1 to 26 in the
preparation of a medicament for the treatment of chronic inflammatory disease.
55. Use of a compound as claimed in any one of claims 1 to 26 in the
preparation of a medicament for the treatment of rheumatoid arthritis,
spondyloarthropathies, osteoarthritis, psoriatic arthritis, or juvenile arthritis,
for the treatment of inflammatory bowel disease, Crohn's disease, ulcerative colitis, or
indeterminate colitis,
for the treatment of psoriasis,
for the treatment of asthma or chronic obstructive pulmonary disease,
for the treatment of stroke, ischemia, or reperfusion injury,
for the lowering of cholesterol, triglycerides, Lp(a), and LDL levels; for the inhibition or
treatment of hypercholesteremia, hyperlipidemia, cardiovascular disease, atherosclerosis,
acute coronary syndrome, peripheral vascular disease, restenosis, or vasospasm,
for the treatment of Alzheimer's disease, cognitive decline, or senile dementia,
for the treatment of type II diabetes,
or for the treatment of sepsis in a mammal.
56 The invention substantially as herein describe.