Note: Descriptions are shown in the official language in which they were submitted.
~'O 93/21145 J ~ 3 3 3 2 5 PCr/l~i93/03909
DESCRIPTION ~
Non-Steroid Proqesterone_Rece tor Aqonist_~nd :
Anta~onist Compounds And Methods ~ ;
Related Application
This application is a continuation-in-part of
application Serial No. 07/872,710 filed April 21, 19g2,
whose entire disclosure is incorporated herein by
reference.
Field of the Invention
This invention relates to intracellular receptors and
ligands therefor. More ~pecifically, this in~ention
relates to compounds which are non-steroidal progesterone j .
receptor antagonists or agonists, and methods for use of
such compounds or ligands.
I~
Backqround of the Invention
A central problem in eukaryotic molecular biology
continues to be the elucidation of molecules and
mechanisms that mediate specific gene regulation in
response to molecular inducers such as hormones. As part
of the scientific attack on this problem, a great deal of
work has been done in efforts to identify molecular
inducers which- are capable of mediating specific gene
regulation.
Although much remains to be learned about the
specifics of- gene regulation, it is known that certain
small molecule, non-peptide hormones and similarly acting
vitamins and-~itamin metabolites (collectively hereinafter
called "horm~nes-") modulate gene transcription by acting
in concert with intracellular components, including
intrace~lular receptors and discrete DNA promoter enhancer
sequences known as hormone response elements (HREs).
These hormones, acting through, and as ~'ligands" for,
their intracellular receptors, directly regulate hormone-
WO93/21145 3 ~ 3 3 3 2 5 PCT/US93/039
responsive genes (and perhaps other important genes whichare not directly hormone-responsive). Natural ligands for
intracellular receptors are synthesized in the body or may
be taken in as a component of food. It has also been
shown that compounds other than the natural ligands can
act upon intracellular receptors to re;gulate hormone-
responsive genes. For example, some: natural product
derivatives and synthetic compounds ~also function as
ligands for these receptors.
10Intracellular receptors form a class of structu~ally-
related genetic regulators scientists have named "ligand
dependent transcription factors." Regulation of a gene by
such factors requires both the intracellular receptor
itself and a corresponding ligand which has the ability to
selectively bind to the intracellular receptor in a way
that affects gene activity. Until bound by a ligand, the
intracellular receptor is unable to exert an effect on the
gene. Hormone or other ligand molecules in the fluid
surrounding a cell pass through the outer cell membrane by
passi~e diffusion. Once inside the cell, the ligand binds
to specific intracellular receptor proteins, creating a
ligand/receptor complex. The binding of the ligand to its
-~receptor induces a change in the shape of the
intracellular receptor. This conformational change is
_ 25 believed to expose regions of the intracellular receptor
-- -- that permit the intracellular receptor/ligand complex to
bind to a specific subset of genes present in the cell's
DNA in the cell nucleus.
- -The blueprint to build specific proteins is encoded
in the DNA sequence of each gene. This blueprint is
copied in a process referred to as "transcription," to
give rise to the actual template for the production of
specific proteins, messenger RNA or "mRNA". The mRN~ then
moves from the cell's nucleus into the cytoplasm and is
translated, which results in the production of proteins
encoded in the mRNA. Accordingly, a reduction in the
~093/~1145 ~1 3 3 3 2 5 PCT/US93/03909
transcription of mRNA reduces the production of the
specific proteins.
Once the intracellular receptor/ligand complex binds
to the specific site on the DNA, it alters the amount of
5 the protein encoded by the gene that the cell is directed `
to produce, by altering the amount of mRNA transcribed by
that gene. A ligand which binds an intracellular receptor
and mimics the effect of the natural ligand is referred to
as an ~agonist" ligand. A ligand that inhibits the effect -
of the hormone is called an "antagonist.~' Intracellular
receptors are referred to as "ligand-dependent transcrip-
tion factors" because their activity is dependent upon the `~
binding of their hormonal or other ligands, which are
necessary to drive the intracellular receptor into its
15 active conformation. j`
~ The intracellular receptors form a large family of
proteins that are closely related in structure. They are
important drug targets, and many drugs currently on the
market are ligands for these receptors. Not surprisingly, j
the structural similarity of the receptors often resultsin cross-reactivity between a drug and receptors other
than its target. It is apparent, therefore, that there
is a need t~ find- alternative ligands (agonists and
antagonists) which are readily available for therapeutic
administration, have_ added specificity for particular
receptors, and have-increased activity. `
Ligands to the progesterone receptor are known to
play an important role in gynecological medicine, cancer,
and other healt-h-care problems of women. Its natural
ligand, the female steroid progesterone, and synthetic
analogues are, _ for example, used in birth control
. _ . .
formulations. -~tagonists to progesterone are useful in
treating chronic disorders such as certain forms of
hormone dependent cancer of the breast, ovaries, and
endometrium ~the lining of the uterus), and in treating
uterine fibroids. Endometriosis, a leading cause of
infertility in women, currently treated in early stage
Wo93/2114~ ;~ L 3 3 ~ 2 5 PCT/US93/03$
development by surgery, is also amenable to treatment with
progesterone.
The identification of compounds which interact with
progesterone receptors, and thereby affect transcription
of genes which are responsive to progesterone, would be of
significant value, e.a., for therapeutic ap~lications such
as treatment of hormonally-responsive gy~ècological and
malignant disorders. -
Further, the identification of compounds which have ;
good specificity for the progesterone receptor, but whichhave lecs cross-reacti~ity for other intracellular
receptors, would be of significant value since interaction
of a ligand with other than the target intracellular
receptors is known to result in significant undesira~le
lS pharmacological side effects. Accordingly, agonists and
antagonists to the progesterone receptor which do not
display cross-reactivity with other intracellular
receptors will exhibit an improved therapeutic index.
A group~of prenylated bromohydroquinones, called col-
leatively cymopols, has been isolated and identified by
; several investigators using as a starting material the-~ ~ green marine alga Cymopolia barbata (L.) Lamouroux
(Dasycladaceae). Among these, cymopol, Cl6H2lBrO2, is a
crystalline phenol which has a bromogeranyl-hydroquinone
2~5~ ~or~brominated~monoterpene-~uinol structure. As described
~~ Hogberg~et al., J.C.S. Perkin I, 1696-1701 (1976),
~`cymopoI ~ ~2-bromo-5-(3,7-dimethylocta-2,6-dienyl~
hydroquinone] and its monomethyl ether, Cl7H23BrO2, ha~e the
$ollowing structures:
~ OH
Br
- OH
- ~ Cymopol
~ ~ .
v093/2ll45 ~ 3 3 2 ~ PCT/US93/03909
. .
S .
.
OMe
Br
OH
'..,~
~ymopol monomethyl ether
Cyclocymopol [l-bromo-3-(4-bromo-2,5-dihydroxy-
benzyl)-2,2-dimethyl-4 methylene cyclohexane] and its
5 monomethyl ether have also been obtained from C. barbata. :~
See Hogberg et al., su~ra. As described in McConnell
et al., Phytochemistry, Vol. 21, No. 8, pp. 2139-41 ¦ ,
(1982), C. barb.ata contains a mixture of optically active ' `
diastereomers of cyclocymopol, Cl6H20Br202, and cyclocymopol
monomethyl ether, C17H22Br2O2, having the following
structures: I :
~. ~
- OR
~ t
, :;
,. .
la (R-Hl. 2a ~R-Me): H (C-3)-pseudo-equatorial
lb (~-H) -2b (R=Me): H (C-3)-pseudo-axial
WO93/21145 ~ 1 3 3 3 2 5 PCT/US93/039~
(The above assumes the equatorial conformation for bromine
at
C - 1 ) .
Through silica gel chromatography of an ether-soluble
extract of C. barbata, McConnell et ai~. were able to
obtain a l:l mixture of ~:~ epimer~^ of cyclocymopol.
McConnell et al. also obtained a 3-:l mixture of ~:~
epimers of cyclocymopol monomethyi ether, which was
enriched to a 4:l mixture of the ~:~ epimers through
purification techniques.
Wall et al., J Nat. Prod., Vol. 52, No. 5,
pp. 1092-99 (1989), described additional diastereomeric
cymopol compounds (cymobarbatol and 4-isocymobarbatol)
which were determined to be hishly active antimutagens.
Wall et al. reported obtaining pure cymobarbatol
compounds, but were unable to obtain stable cyclocymopol
fractions. Apparently, however, the forms of cyclocympol
and cyclocymopol monomethyl ether obtained by Hogberg et
al., su~ra, were pure forms of formulae lb and 2b above.
The publications and references ref~rred to above and
hereafter in this specification are incorporated herein by
reference.
SummarY of the Invention
- _ The present invention is directed to compounds,
~~ -2-5 --compo~itions, and methods for modulating processes
- mediated by progesterone receptors. More particularly,
the invention relates to non-steroidal compounds which are
~ ~- high affinity, high specificity ligands for progesterone
- - receptors. These compounds exhibit progesterone receptor
_-- 30 agonist or progesterone receptor antagc~ist activity, and
-~- - modulate processes mediated by progesterone receptors.
Accordingly, the invention also relates to methods for
modulating processes mediated by progesterone receptors
employing the compounds disclosed. Examples of compounds
used in and forming part of the invention include
cyclocymopol derivatives and purified diastereomers
WO93/21145 ~ 1 3 3 3 2 .j PCT/US93/03909
thereof, synthetic cyclocymopol analogs, and semisynthetic
derivatives of natural cyclocymopols. Pharmaceutical
compositions containing the compounds di~closed are also
within the scope of this invention. Also included are
methods for identifying or purifying progesterone
receptors by use of the compounds of this invention.
Brief Descr,iption of the Fiqures
The presen~ invention may be better understood and
its advantages appreciated by those skilled in the art by
referring to the accompanying drawings wherein:
Figure 1 presents the proton NMR spectrum for the
individual pure 3R (panel a) and 3S (panel b)
diastereomeric acetates of cyclocymopol monomethyl ether.
Figure 2 presents activation profiles for analysis of
progesterone receptor activation by a cyclocymopol
monomethyl ether diastereomeric mixture (compound S0-44),
by a pure 3S diastereomeric acetate (compound SO-51), and
by a pure 3R diastereomeric acetate (compound S0-52). For
these compounds and a progesterone control, agonist dose
response is shown in panel a, and antagonist dose response
in panel b. -
Figure 3-~resents activation profiles for analysis of
progesterone receptor activation by (3R)-cyclocymopol
monomethyl ether (compound S0-53). For this compound and
a progesterone control, agonist dose response is shown in
panel a, and antagonist dose response is shown in panel b.
, Figure 4 presents activation profiles for analysis of
progesterone receptor activation by (3S)-cyclocymopol
monomethyl e~her (compound S0-54) and its acetate
(compound SO-51)-.-,,For these compounds and a progesterone
control, agonist-- dose response is shown in panel a, and
antagonist dose re ponse is shown in panel b.
Figure~5 presents acti~ation profiles for analysis of
progesterone receptor activation by (3R)-cyclocymopol
3s monomethyl ether (compound S0-9). For this compound and
WO93/2114S `~l 3 3 ~ 2 5 PCT/US93/039~
a progesterone control, against dose response is shown in
panel a and antagonist dose response is shown ln panel b.
Figure 6 presents activation profiles for analysis of
glucocorticoid receptor activation by (3R)-cyclocymopol
monomethyl ether (compound SO-09). For this compound and
a dexamethasone control, agonist dose ~esponse is shown in
panel a and antagonist dose response is shown in panel b.
Figure 7 presents profiles of displacement of 3H-
labeled progesterone by cyclocymopol monomethyl ether
diastereomers (panels a and b), and of the 3H-labeled
progesterone agonist R5020 by RU486 and by a (3R)-
cyclocymopol monomethyl ether compound (SO-9).
Figure 8 presents profiles for analysis of
progestrone binding for RU486 and (3R)-cyclocymopol
monomethyl ether (compound SO-9).
Figure 9 presents profiles of the displacement of 3H-
labeled dexamethasone from gIucocorticoid receptor for
several compounds.
Figure lO presents profiles showing the functional
activities of cyclocymopol analogues in T47D cells. Panel
a shows ligand dependent induction of alkaline phosphatase
in T47D cells by RU486 and cyclocymopol monomethyl ether
diastereomers. Inhibition by (3R)-cyclocymopol monomethyl
ether (S0-53) of progesterone-stimulated induction of
- - Z`5~ alkaline phosphatase is shown in panel b, and of R5020
~- ~ ~~stimulated induction in panel c.
Figure ll presents profiles showing the inhibition by
RU486 of induction of alkaline phosphatase in T47D cells
by (3S)-cyclocymopol monomethyl ether (S0-54) in panel a
and by its acetate (SO-51) in panel b.
_
--- -
Detailed Descri~tion of the Preferred Embodiment
Cyclocymopols useful in this invention are defined as .
those having the formulae:
~093J21145 ~ ~ 3 3 ~ 2 5 PCT/VS93/03909
~'
R R ~ R
4 5
or
R R 1~ R
wherein: - -
the dotted lines in the structure depict optional I `
double bonds;
X is carbo~, oxygen, or nitrogen;
Rl is Rl7, -ORl"--N(R17)(Rl7), -SR17, fluorine, chlorine,
bromine, or -NO2;
, ~ ,
Rl7 and- (R17-), each independently, are hydrogen,
saturated or unsaturated Cl-C6 alkyl, C3-C7 cycloalkyl, Cs-C7
aryl, or C7 aralkyl, said alkyl groups being branched or
straight-chain;
R2 is -NO2, -N(OH)Rl7, fluorine, chlo~ine, bromine,
iodine, Rl7, -N(Rl7)(Rl7,), -SRl7, -S(O)-Rl7, -S(O)2-Rl7,
CH2OH, -C~O)-H, -C(O)CH3, -C(O) -OCH3, -C=CH2, -C=CH-C(O)- I :
OCX3, or Rl8;
~ 1 3 3 ~ 2 ~ PCT/US93/03~
Rl8 and (Rl8), each independently, are hydrogen,
saturated or unsaturated C1-C6 alkyl, C3-C6 alkyl, C3-C7
cycloalkyl, Cs-C7 aryl, or C7 aralkyl, said alkyl groups
being branched or straight-chain which optionally may
contain hydroxyl, aldehyde, ketone, nitrile, or ester
groups;
R3 is Rl7 or -OR17;
R4 is hydrogen, -OR1" -OC(O)R17, -OC(O)OR17,
-OC(O)N(R17)(R17), -OS(O)2R17, or -OS(O)-R17;
R5 is hydrogen or ORl7;
R6 is R17;
R7 and R3, each independently, are Rla, or R7 and R8
together are a carbocyclic 3-8 member ring;
Rg and Rlo, each independently, are chlorine, bromine,
or R17, or R9 and Rlo combined are =0, except when X=0, Rg
and Rlo are not present, and when X is N, then Rlo iæ not
present, or Rg and Rlo together are joined in a carbocyclic
3-8 member ring;
~ Rll and R12, each independently, are -ORl7, Rl8, are =0,
or are =CH2 except when Rl1 is attached to an sp~ carbon
atom in the ring, then Rl2 is not present and Rll is Rla,
or R1l and Rl3 together are joined in a carbocyclic 3-8
- member ring or are -O- to form an epoxide;
Rl3 and R14, each independently, are -OR17 or R1a, except
when R13 is attached to an sp2 carbon atom in the ring, then
: - -- Rl4 is not present and Rl3 is -ORl, or Rl8;
Rls and Rl6, each independently, are R1a or OR1~, or Rl5
and Rl6 together are -CH2-O- forming an epoxide, or Rls and
~ - Rl6 combined are =0 or =C(Rla)(Rla), except when Rls is
hydroxyl, then Rl6 is not hydroxyl, and when Rls is attached
-- to an sp2 carbon atom in the ring, then R16 iS not present.
,
~093~2114~ 3 3 ~ a PCT/US93/039
11
Definitions
In accordance with the present invention and as used
herein, the following terms axe defined with the following
meanings, unless explicitly stated otherwise.
The term alkyl refers to straight-chain, branched-
chain, cyclic structures, and combinations thereof.
The term "aryl" refers to aromatic groups which have
at least one ring having a conjugated pi electron system
and includes carbocyclic aryl, heterocyclic aryl and
biaryl groups, all of which may be optionally substituted,
being preferably phenyl or phenyl substituted by one to
three substituents, such substituents being advantageously
lower alkyl, hydroxy, lower alkoxy, lower acyloxy,
halogen, cyano, trihalomethyl, lower alcylamino, or lower
alkoxycarbonyl.
Carbocyclic aryl groups are groups wherein the ring
atoms on the aromatic ring are carbon atoms. Carbocyclic
aryl groups include monocyclic carbocyclic aryl groups and
optionally substituted naphthyl groups.
Heterocyclic aryl groups are groups having from l to
3 heteroatoms as ring atoms in the aromatic ring with the
remainder of the ring atoms being carbon atoms~ Suitable
heteroatoms- include oxygen, sulfur, and nitrogen, and
suitable heterocyclic aryl groups include furanyl,
thienyl, pyridyl,_ pyrrolyl, N-lower alkyl pyrrolyl,
pyrimidyl, pyrazinyl, imidazolyl, and the like, all
optionally su~stituted.
The term "aralkyl'l refers to an alkyl group
substituted-with-an aryl group. Suitable aralkyl groups
include benzyl and the like, and may be optionally
:~.
substituted. _
The term "lower" referred to herein in connection
with organic radicals or compounds respectively defines
such wi~h up to and including 7, preferably up to and
3~ including 4 and advantageously one or two, carbon atoms.
Such groups may be straight chain or branched.
WO 93/21145PCT/US93/~34
2 l 3 3 3 2 5
12
Representative compounds and derivatives according to
the present invention include the following:
1- Methyl idene - 2 - ( 2 ' - acetoxy- 4 ' -bromo - 5 ' -
methoxyphenyl ) methyl - 3, 3 -dimethylcyclohexane;
5(3S) -l-Debromocyclocymopol monomethyl ether, 2 ' -
acetate;
l-Methylidene-2- (2 ' -hydroxy-4 ' -bromo-5 ' -
methoxyphenyl ) methyl - 3, 3 - dimethylcyclohexane;
(3S) -ï-Debromocyclocymopol monomethyl ether, 2 ' -
methylcarbonate;
(3R, 5R) -5-Hydroxycyclocymopol monomethyl ether; 2-
(4 ~ -Nitrophenyl) methylcyclohexanone;
( 3S) -1 -Debromocyclocymopol monomethyl ether;
l-Methylidene-6 - (2 ' -acetoxy-4 ' -bromo- 5 ' -
methoxyphenyl)methyl-3,5,5-trimethylcyclohex-2-ene;
l-Methylidene-6- (4' -nitrophenyl)methyl-3, 5, 5-
trimethylcyclohex- 2 -ene;
( 3R) -1 -Debromocyclocymopol monomethyl ether;
l-Methylidene-6- (2 ' -hydroxy-4 ' -bromo-5 ' -
2 0 me thoxyphenyl ) methyl - 5, 5 - dimethyl cyc lohex- 2 - ene;
l-Methylidene-6- (2' -acetoxy-4' -bromo-5' -
methoxyphe~yl ) methyl - 5, 5 -dimethylcyclohex- 2 -ene;
l-Methylidene-6- (3 ' -methyl-4 ' -nitrophenyl) methyl-5, 5-
dimethylcyclohex-2-ene;
-~ -~~ 25 trans-1-Methylidene-6- (2 ' -acetoxy-4 ' -bromo-5 ' -
methoxyphenyl)methyl-4, 5, 5-trimethylcyclohex-2-ene;
1 -Methyl idene - 2 - ( 4 ' -bromophenyl ) methyl - 3, 3 -
~- ~ dimethylcyclohexane;
. . .
1- Me thyl idene - 2 - ( 2 ' - hydroxy - 4 ' -bromo - 5 ' -
- 30 methoxyphenyl)methyl-3,3-dimethylcyclopentane;
- ~ l-Methylidene-2- (4 ' -nitrophenyl) methylcyclohexane;
( 3R) - l-Debromocyclocymopol monomethyl ether, 2 ' -
methylcarbonate;
1 - Me t hy 1 i de ne - 2 - ( 2 ' - hydroxy - 4 ' - bromo - 5 ' - me t hoxy - ¦ .
35 phenyl ) methylcyclohexane;
(3R)-Cyclocymopol monomethyl ether,2'-methylcarbonate;
I;
21333~5
V~93/21145 PCT/US93/03909
.
13
(3R)-l-Debromocyclocymopol monomethyl ether,2~-
benzoate; and
~ 3R)-4'-Iodocyclocymopol monomethyl ether.
Compounds comprising the class of cyclocymopol
compounds and derivatives disclosed herein can be obtained
by routine chemical synthesis by those skilled in the art,
e.g., by modification of the cyclocymopol compounds
disclosed or by a total synthesis approach.
The cyclocymopol compounds of this invention bind
selectively to the progesterone receptor. We have found
that the non-synthetic cycloc~mopol compounds have agonist
or antagonist acti~ity depending on their stereoisomeric
form. For example, the 3~ or 3R diastereomer of
cyclocymopol monomethyl ether has progesterone receptor
antagonist activity, and the 3~ or 3Sdiastereomer of
cyclocymopol monomethyl ether has progesterone receptor
agonist activity. In contrast, other cyclocymopol analogs
or derivatives have been found to predominently exhibit
progesterone receptor antagonist activity regardless of
their stereoisomeric form.
In the current invention, individual diastereomers of
cyclocymopol monomethyl ether have been isolated from each
other and~~purif-ied in accordance with the following
example.
.
Exam~le l ~ `-- -
The marine- alga Cymopolia barbata (L.) Lamouroux
! ' (Dasycladaceae) was collected and frozen. Frozen sample
was lyophilizëd~ and extracted with l:l MeOH/CH2Cl2 three
times, and the extract was concentrated n vacuo to obtain
an a~ueous~ suspension of organic components. The
concentrate-~as re-extracted with CH2Cl2 until no color
came into the organic phase, and the CH2Cl2 extract was
then concentrated to obtain the crude extract as a dark,
green oil. The crude extract was purified by column
chromatography on Sephadex LH20 with l:l MeOH/CH2Cl2, or
vacuum flash chromatography on silica using a gradient of
W093/2114~ PCT/US93/03~
'~13332~ 14
ethyl acetate in hexane, and the fractions were examined
by thin layer chromatography ~TLC). The fractions that
contained cymopols were pooled together and separated by
reversed pha~e high performance liquid chromatography
s (HPLC) using 80% MeOH/H20 to yield a mixture of
cyclocymopol monomethyl ether diastereomers which were
identified by nuclear magnetic resonance (NMR) spectro-
scopy as a mixture of the following as shown below:
OMc OMe
O~H ~ ~ Br
Cyclocymopol monomethyl ether diastereomers
Proton NMR spectrum of the cyclocymopol monomethyl
`~ ether mixture indicated the presence of the two
diastereomers in the ratio 4:1. The two compounds could
not be separated by reversed phase HPLC (ODS Column, 20~
H20/MeOH solvent) where both migrated with the same
- ; retention.
The two compounds behaved similarly on normal phase
HPLC (silica column, 5% EtOAc/hexane solvent), and an
- attempt to collect fractions inside the HPLC peak also
failed to yield any separation. This indicated that the
diastereomers of the mixture had very similar
~-~ chromatographic behavior, even though diastereomers
normally have different chromatographic characteristics.
~ ! l In order to separate the diastereomèrs, the
-~ 25 cyclocymopol monomethyl ether diastereomer mixture was
- ~ reacted with acetic anhydride (Ac20) and pyridine at room
, . . .
temperature for about 10 hours. The resulting mixture of
diastereomaric acetates was completely separated by normal
phase HPLC (silica column, 5% EtOAc/hexane) to yield the
individual chromatographically-pure diastereomers, as
follows:
, .
:
WO93/21145 ~ 3 3 3 2 ~ PCT/US93~03909
OMe OMe
~Br ~Br
OAc ~ OAc ~ ;
Diastereomeric Acetates
Proton NMR analysis confirmed that two separate
pectroscopically-pure diastereomeric acetates were
obtained, as shown in Figure 1. The spectrum for t:he 3R
diastereometric acetate is shown in panel a, and the
spec~rum for the 3S diastereometric acetate is shown in
panel b of Figure 1. The individual pure diastereomers
were separately reacted with K2CO3 in MeOH to conver~ them
back to the parent form, and the resultant products were
purified by re~ersed phase HPLC using 80~ MeOH/~2O to
obtain the individual purified diastereomers of .
cyclocymopol monomethyl ether, as follows: i
OMe OMe
O~ ~ ~ Br
.. -. .
3~ or 3R 3~ or 3S
diastereomer diastereomer
Proton NMR analysis of the resulting indi~idual .
cyciocymopol monomethyl ether diastereomers also showed
them to be spectroscopically pure. The 3R or 3
diastereomer was determined to ha~e been the major
component of the cyclocymopol monomethyl ether
diastereomer mixture obtained from C. barbata.
WO93/2114~ PCT/US93/039~
213332~
Proqesterone RecePtor Activity
Utilizing the "cis-trans" or "co-transfection~ assay
described by Evans et al., Science, 240:889-95 (May 13,
1988), the two cyclocymopol monomethyl ether diastereomers
s were tested and found to have actlvity specifically for
the intracellular receptor for progesterone. This assay
is described in further detail in U.S. Patent
Nos. 4,981,784 and 5,071,773, which are incorporated
herein by reference. The co-transfection assay provides
a method for identifying functional ligands (either
agonists which mimic, or antagonists which inhibit, the
effect of hormones) for ligand-responsive receptor
proteins.
The co-transfection assay provides a mechanism to
evaluate ability of a compound to function as an agonist
or antagonist of the activity modulated by an
intracellular receptor.- The co-transfection assay mimics
an ~n vivo system in the laboratory. In the co-
transfection assay, a cloned gene for an intracellular
receptor is introduced by transfection (a procedure to
induce cells to take up foreign genes) into a background
- cell substantially devoid of endogenous intracellular
receptors. This introduced gene directs the recipient
cells to make the intracellular receptor protein. A- - 25 second gene is a}so introduced (co-transfected) into the
same cells in conjunction with the intracellular receptor
gene. This second gene functions as a reporter for the
! itranscription-modulating activity of ~he target
intracellular receptor. The reporter acts as a surrogate
for the products normally expressed by a gene under
control of the target receptor and its natural hormone.
A preferred reporter gene is one which expresses the
firefly enzyme luciferase. The co-transfection assay can
detect small molecule agonists or antagonists of target
intracellular receptors. Exposing the cells to an agonist
ligand increases reporter activity in the transfected
cells that can be conveniently measured, reflecting
':?:
'~
VO93/21145 ~ ~ 3 ~ 3 2 S PCT/VS93/03909
ligand-dependent, intracellular receptor-mediated
increases in reporter transcription. To detect anta-
gonists, the co-transfection assay is carried out in the
presence of a constant concentration of an agonist known
to induce a defined reporter signal. Increasing
concentrations of a test antagonist will decrease the
reporter signal. The co-transfection assay is therefore
useful to detect both agonists and antagonists of specific
intracellular receptors. It determines not only whether
a compound interacts with a particular intracellular
receptor, but also whether this interaction mimics
(agonizes) or blocks (antagonizes) the effects of the
natural regulatory molecules on target gene expression.
Co-transfected cells are-exposed to a medium to which
is added the potential ligand that is being evaluated. If
the candidate ligand diffuses into the cell and binds to
the receptor and the resulting complex functions as an
agonist, it binds to the co-transfected reporter gene and
initiates transcription. When that gene is one that
expresses, for example, luciferase, luciferase is produced
which catalyzes a light-emitting reaction with its
substrate- luciferin. Thus, after cell lysis and the
introduc~ion of luciferin, the amount of light produced
relative to the concentration of candidate ligand used in
the assay provides a measure of the potency and efficacy
of the compound tested. Antagonist activity is evaluated
by adding the-candidate ligand and a known agonist to the
coj-transfected cells. Suppression of agonist-induced
luclferase-production by the candidate compound, and hence
the amount of light produced, indicates the candidate
ligand ls--~n antagonist.
The -progesterone receptor activity of the cyclo-
cymopol monomethyl ether disastereomer compounds were
demonstrated according to the following illustrative
example.
EDIT OPERATOR, PLEASE TYPE THIS PAGE.
~ O 93/21145 2 1 3 3 3 2 ~ PCT/US93/03909
19 .~:
also shown in Figure 2, the corresponding purified 3R
diastereomeric acetate (designated S0-52) exhibited
progesterone receptor antagonist activity, and a purified
3S diastereomeric acetate (designated S0-51) exhibited
progesterone receptor agonist activity. These assays also
showed that a 3R diastereomer of cyclocymopol monomethyl
ether (designated S0-53) exhibited progesterone antagonist
activity, as shown in Figure 3. In contrast, the 3S
diastereomer of cyclocymopol monomethyl ether (S0-54), and
its acetate (S0-51), display progesterone receptor agonist
ac~ivity, as shown in Figure 4.
Compound S0-9 and the identical compound from a
separate preparation, compound S0-53, exhibit progesterone
receptor antagonist activity (see Figure 5), and were
tested for cross reactivity wi~h the other known
intracellular receptor classes, e.g., glucocorticoid,
mineralocorticoid, androgen, estrogen, and retinoic acid.
Compound S0-9 was also tested with orphan receptors (which
are receptors whose natural ligand is unknown). The
compound was found not to cross-react with any of the
other receptors, which demonstrates that activity was
limited to the progesterone receptor. Figure 6 is
illustr-ative, and shows that compound S0-9 demonstrated
neither agonist nor antagonist activity with the
glucocorticoid receptor, dexamethasone.
The 3R and 3S diastereomeric acetates of cyclocymopol
monomethyl ether were also individually tested for cross
reactivity with the other known intracellular receptor
classes. This testing showed the 3R diastereomer to have
slight agonist activity with the glucocorticoid receptor.
No antagonist activity was detected with either compound.
To investigate the interaction of the cyclocymopol
analogs with the human progesterone receptor, and to
further demonstrate that they display ligand activity with
the progesterone receptor, the analogs were tested for
their ability to displace 3H-labeled progesterone from cell
WO93/21145 PCT/US93/039
2133~25
extracts containing the human progesterone receptor. ;
These tests were conducted according to the following ;~
illustrative example.
Exam~le 3
A plasmid which expresses progesterone receptor was ;
transfected into CV-1 cells by the method of calcium
phosphate precipitation. After six hours, the cells were
washed and incubated at 37C with 95~ 2/5~ C2 for 40
hours prior to harvest.
After incubation, cells were harvested and washed in
phosphate buffered saline. Whole cell receptor extract
was prepared by homogenizing the harvested cells in Tris-
HCl buffer, pH=7.4, containing 30% glycerol, 1 mM EDTA, 12
mM monothioglycerol, 1 mM PMSF, and 0.5 M potassium
chloride. The homogenate was incubated at 4C for 60 min
with resuspension every 10 min. The suspension was
centrifuged (105,000 X g, 60 min) and the supernatant was , 1;
collected and flash frozen in liquid nitrogen and stored ! -`
frozen at -70C.
Aliquots of the whole cell extract containing .-
- - transfected progesterone receptor was incubated at 4C for
24 hours with a constant conc~ntration (5 nM) of tritiated
progesterone and increasing concentrations (0 - 2.5 x 10- 1
- _ sM) of either unlabeled cold progesterone or test compound.
25- The concentration of bound tritiated progesterone was
i
determined in each sample by the dextran-coated charcoal I
adsorption technique, as follows.
_ ~ ~~ To a 500 1 final volume incubation mixture, 400 1 of -
- 7.S% (w/v) dextran-coated charcoal suspension in gelatin
phosphate buffer was added. The mixture was vortexed and
- ~ -- incubated at 4C for 10 min and then centrifuged at 3000
rpm for 10 min. The amount of bound tritiated hormone was `
determined by liquid scintillation spec~rophotometry of an
aliquot of the supernatant.
WO93/21145 ~1 3 3 3 2 5 PCT/US93/03909
21 ;`
As shown in Figure 7a, the purified 3R diastereomer
of cyclocymopol monomethyl ether (compound S0-53) and its
corresponding acetate (compound S0-52) displaced 3H-
progesterone from its ligand binding site in a
concentration-dependent manner. Similar binding isotherms
were obtained with the purified 3S diastereomeric acetate
of cyclocymopol monomethyl ether (compound SO-51), as
shown in Figure 7b. These compounds were between 2 and 3
orders of magnitude less potent than the endogenous
hormone, progesterone. Similar binding studies were also
carried out using the radiolabeled progesterone-agonist 3H-
R5020, compound SO-9 (which is identical to compound SO-
53), and the progesterone antagonist, RU486. As shown in
Figure 7c, RU486 was a competitive antagonist of R5020,
whereas compound SO-9 did not compete for this binding
site.
.
~Progesterone receptor antagonists, such as RU486, can
- ~increase the specific binding of progesterone to its
ligand binding site. This enigma was also observed upon
performing binding studies with 3H-progesterone in the
presence of compound SO-9. As shown in Figure 8, this
compound signi-ficantly increased the apparent Bmax of 3H-
progesterone. ~
Specificity of binding of progesterone agonists and
25 ~antagonists has been studied by comparing the ability of
various ligands t~- displace 3H-glucocorticoid from the
human glucocorticoid receptor. The data in Figure 9a
lustrate that each of the ligands studied has affinity
for the glucocort-icoid receptor. The affinity of RU486
for the glucocorticoid receptor exceeds that of
dexamethasone, -and is 300-fold greater than that of
compound SO-09-~.--=In fact, the affinity of RU486 for the
glucocorticoid receptor is comparable to its affinity for
the progesterone receptor.
.
35The functional activities of the cyclocymopol
analogues in the human breast cell line, T47D, were also
WO93/21145 PCT/US93/039~
'~ 133325
22
investigated. T47D cells have proven to be a particularly
useful model to investigate the molecular actions of sex
steroids because they contain endogenous functional
receptors for, and respond to, progestins, and their
respecti~e antagonists. Moreo;~er, these cells contain
exceptionally high titexs of progesterone receptors and
are exquisitely sensitive to the actions of progestins in
a manner quite similar to their actions in normal and
neoplastic mammary epithelial cells. In T47D cells,
progestins induce de novo synthesis of a plasma-associated
alkaline phosphatase, which has been reported to be
similar, if not identical, to the alkaline phosphatase
present in the normal breast and human milk. These te~sts
were conducted according to the following illustrative
example.
I ~,
Exam~le 4
T47D cells were cultured in RPMI 1640 medium
ortified with 10~ fetal bovine serum, 2 mM glutamine, 0.2
ug/ml bovine insulin and, 0.05 mg/ml gentamicin. Cells
were plated in 100-mm plates in medium; 4~ hours later
- they were changed to medium containing 2~ charcoal treated
~ -- serum with or without test compounds in a final ethanol
concentration of 0.1%. For routine induction of alkaline
.._phosphatase, cells were treated for three days with two
~-25 -media changes and harvested as described below.
- Cells were collected with a rubber policeman into
p~osphate-buffered saline, pelleted, and lysed with TPSG
~ ~ - .buffer (0.2% Triton X-100 containing 10 mM sodium
- phosphate pH-7.4, 0.1 M sucrose, and 10% glycerol) at 0
C for 30 min with vigorous vortex mixing every 5 min.
- ~- Nuclei were sedimented at 2500 rpm, and the supernatant
was saved as cyto901. The protein content of the cytosol
~ was assayed by the method of Bradford. Alkaline
phosphatase activity was determined by incubating one
3s volume of cell extract with three volumes of 1 mg/ml p-
nitrophenol phosphate pr~epared in DEAM (l M
WO93/2114~ 3 ~ 3 2 ~ PCT/US93io3909
diethanolamine, pH=s~ containing 2 mM magnesium chloride)
at room temperature for 20 min. At the end of the
incubation time, the reaction was stopped with an equal
volume of lN NaOH. Ten standards of p-nitrophenol were
~ 5 prepared in DEAM buffer and absorbance was read at 405 nm.
Alkaline phosphatase acti~ity is expressed as pmol p-
nitrophenol formed/min/mg protein.
As shown in Figure l0a, (3S)-cyclocymopol monomethyl
ether (compound S0-54), and its corresponding acetate
(compound SO-5l), are functional agonists in this system,
whereas (3R)-cyclocymopol monomethyl ether (compound SO-
53), and its corresponding acetate (compound S0-52),
appear to be very weak agonists. The 3S form compounds
exhibit increased efficacy at higher concentrations, an
effect which has been found to be reproducible.
To detexmine if (3R)-cyclocymopol monomethyl ether
could function as a progesterone receptor antagonist, the
effects of increasing concentrations of this compound on
R5020-induced alkaline phosphatase activity were
quantified in T47D cells. As shown in Figures l0b and
l0c, (3R)-cyclocymopol monomethyl ether (compound S0-53)
is an effective antagonist of the progesterone mimic.
Similarly, when alkaline phosphatase activity was
stimulated by l_~--nM~ ~rogesterone, (3R)-cyclocymopol
monomethyl ether (compound S0-53) attenuated this
induction in a concentration-dependent manner.
In comparison, the progesterone agonist, (3S)-
cyclocymopol monomethyl ether (compound S0-54), and its
acetate (SO-51), increased the activity of intracellular
alkaline phospha~ase~~in a concentration-dependent manner,
and this was blocked by the progesterone antagonist,
RU486, as shown in Figure ll. In this system, (3R)-
cyclocymopol monomethyl ether (compound S0-53) functioned
as a progesterone receptor antagonist and attenuated the
WO93/~114~ PCT/US93/039~
~3332S 24
_. .
effects of progesterone in a concentration- dependant
manner.
Synthetic and semisynthetic cyclocymopol analogs have
been prepared which also have activity for the
intracellular receptor for progesterone. Representative
analogs of the present invention-are prepared according to
the following illustrative synthetic schemes and
illustrative examples.
Exam~le 5 - Svnthesis of Aromatic Subunit
2-AcetoxY-5-methoxYbenzaldehYde ~
To a flame-dried 50 mL round-bottomed flask
containing 10.00 g (6S.7 mmol) 2-hydroxy-5-methoxy
benzaldehyde in 10 mL dry pyridine at 0C under nitrogen
atmosphere was added 7 mL acetic anhydride. The reaction
mixture was then allowed to warm to room temperature and
continually stirred until TLC analysis indicated complete
consumption of starting material (50 min). Ethyl acetate
tl50 mL) was added, and the mixture was then transferred
to a separatory funnel and successively washed with lN HCl
(3 x 50 mL), saturated aqueous NaHCO3 (1 x 50 mL), and
~~brine (1 x 50 mL), dried over Na2S0~, and concentrated
under reduced pressure to give 12.41 g (97~) of the
- w etylated phenol as a white solid. The product thus
-obtained was homogenous by TLC (Rf 0.41, 2:1 hexane/ethyl
acetate), and was carried on to the next step without
~further pur;ification.
~ 2-Acetoxv-4-bromo-5-methoxYbenzaldehyde (2~:
--To a 500 ~L round-bottomed flask containing a
solution of 20.0 g (168.1 mmol, 3.26 equiv) potassium
bromide and 3.21 mL (lO.0 g, 62.6 mmol, 1.21 equiv)
bromine in 200 mL water at room tèmperature was added
- 10.00 g (51.5 mmol, 1.O equiv) 2-acetoxy-5-
methoxybenzaldehyde (1) as a finely divided white powder,
portionwise over a period of 35 min. After 18 h stirring
3S at room temperature, the reaction mixture was filtered
W093/21~4s ~ 3 3 3 2 ~ PCT/US93/03909
under vacuum using a Buchner funnel to give 10.59 g (89~)
of the aryl bromide as a pale yellow solid (Rf 0.45, 2:1
hexanes/ethyl acetate). The product thus obtained was of
greater than 98~ purity by lH NMR, and homogenous by TLC,
and was carried on to the next step without further
purification. A portion of the crude product was
recrystallized from 5:1 ether/hexanes to give white
needles.
2-HYdroxy~-bromo-5-methoxYbenzaldehy~e (33:
To a 200 mL round-bottomed flask containing 6.90 g
(25.3 mmol) 2 acetoxy-4-bromo-5-methoxybenzaldehyde ~2) in
100 mL of 1~ aqueous methanol at room temperature was
added 5 g K2C03, and the mixture was allowed to stir at
room temperature for 1 h, at which time TLC analysis
indicated complete consumption of starting material and
the presence of a slightly more polar compound as the only
detectable product. The reaction mixture was then
neutralized to pH 5 with the addition of 1 N HCl, and the
solvent was subsequently removed under diminished
pressure. The residue was then dissolved in ethyl acetate
(200 mL), washed successively with 1 N HCl (1 x 50 mL),
saturated aqueous NaHCO3 (1 x 50 mL), and brine (1 x 50
mL), dried o~e~ Na2S04, and concentrated under diminished
pressure to give 4.97 g (85%) of the bromophenol as a
brownish-red oily solid. A portion of this crude material
was recrystaIlized:from 2:1 hexanes/ethyl acetate to give
white needles. The r~mainder of the material was carried
on to the next step without further purification.
2-(ten-Butvl)dimethylsilvloxy-4-bromo-5-methoxybenzaldehyde
(4): - -
To a flame-dried 100 mL round-bottomed flask
containing 2.7~--g (11.95 mmol) 2-hydroxy-4-bromo-5-
methoxybenzaldehyde (3) in 50 mL anhydrous dichloromethane
under nitrogen atmosphere at room temperature was added
2.03 g (29.88 mmol, 2.50 equiv) imidazole, 2.25 g (14.94
mmol, 1.25 equiv) (tert-Butyl)- dimethylchlorosilane, and
DMAP (100 mg, catalytic). The mixture was allowed to stir
WO93/21145 PCT/US93/039
'~ 133 32 S 26
at room temperature for 75 min, at which time T~C analysis
indicated complete consumption of starting material, and
the formation of a less polar product ~Rf 0.75, 2~
hexanes/ethyl acetate). The reaction mixture was then
poured into a separatory funnel : containing 50 mL
dichloromethane and 50 mL saturated a~ueous NX4Cl, the
layers were separated, and the organic phase was washed
with 50 mL brine, dried over Na2SOç, and concentrated under
diminished pressure to give 4.13 g (quantitative) of the
silylated bromophenol as an off-white solid, a portion of
which was recrystallized from 3:1 hexanes/ether to give an
amorphous white solid.
2-(tert-Butvl ~dimethylsilyloxv=4-bromo-5-methoxybenzYl
alcohol (5):
15To a flame-dried 200 mL round-bottomed flask
containing 4.10 g (13.14 mmol) 2-(tert-butyl)dime~
thylsilyloxy-4-bromo-5-methoxybenzaldehyde (4) in 100 mL
anhydrous methanol at 0C was added 0.50 g ~13.15 mmol,
1.0 mol equiv) NaBH4 over a period of 3 min. After 20 min
at OoC, TLC analysis indicated complete consumption of
starting material, and the formation of a more polar
--- product (Rf 0.42, 2:1 hexanes/ethyl acetatel. Wa~er (75
mL) was added, and the methanol was removed by rotary
_ evapora-tion. The resultant aqueous residue was extracted
- 25 with ethyl acetate (2 x 100 mL), and the combined organic
~ layers were dried over Na2SO4, and concentrated under
diminished pre sure. Purif-ication by flash column
~ chromatography (silica gel, hexanes/ethyl acetate, 5:1)
- afforded 4.10 g (98%) of the benzylic alcohol as a white
solid.
. .
_2-(tert-ButYl)dimethYlsilyloxy-4-bromo-5-methoxYbenzvl
bromide (6):
To a flame-dried 100 mL round-bottomed flask
containing triphenylphosphine (1.27 g, 4.84 mmol, 1.05
equi~) in 25 mL anhydrous DMF at 0C under nitrogen
atmosphere was added bromine (0.24 mL, 4.84 mT, 1.05
wo 93/2114~ ~13 ~ 3 2 S Pcr/us93/03909
equiv, plus enough extra to cause a persistent reddish
tint to the solution, 1 drop) through an additional
funnel. To this reaction mixture was added 2-(te~-
butyl)dimethylsilyloxy-4-bromo-5-methoxybenzylalcohol(5)
through the addition funnel at a steady rate over 30 min,
as a solution in 10 mL DMF. The reaction mixture was
allowed to stir at 0C for 60 min, at which time TLC
analysis indicated complete consumption of starting
material, and the formation of a less polar product (R~
0.81, 2:1 hexanes/ethyl acetate). Hexane (100 mL) was
then added, and the contents of the flask were trans-
ferred to a separatory funnel containing 50 mL of
saturated aqueous NH4Cl, rinsing with an additional 50 mL
hexane and 10 mL water. The layers were separated, and
the organic phase was washed with 20 mL 10% Na2S203, dried
over Na2SO4, and concentrated under diminished pressure. 1~
Purification by trituration at 0C (2 x 30 mL ea. hexane) :
to remove residual triphenylphosphine oxide, followed by
flash column chromatography (silica gel, hexane/ethyl ¦ :
acetate, gradient elution) afforded 1.51 g (80%) of the
benzylic bromide as a colorless, viscous oil. l~ NMR (400
MHz, CDCl3) -~ - 0.28 [s, 6H, Si(CH3)2], 1.05 [s, 9H,
SiC~CH3)3], 3.87-(9,-3H, OCH3), 4.48 (s, 2H CH2Br), 6.79 and
7.01 ppm (2s, 2 x lH, Ar-H).
.
_ - -
. -- - .
WO 93/21 l452 1 3 3 3 2 ~ PCr/US93/039~
,
28 -
iOMe OMe
Ac~O / pyridine ~ Br2 / K~r / H~O
I , .- . . _ I
~,H n/50m-n ~ n/18h
OMe OMe
ar~ S%K2t::O3 /MeOH 3r~Tf~ SCI/~nidazole . -~
_ ~ H ~ i
~H rt/lh ~f CH2Cl2JDMAP(cat~ i
OA~: O 85% OH o 100%
2 3 :
OM~ OMe ..
E~r~ N~BH~ / MeOH Br~q .
~ 0C 120min ~ OH
TE3SO o TBSO
4 5 `
.,_,
-, ,
OMe
Ph3~ / Br2 3r,l~
`1
DMP10C ~Br -
8i~% 1 .
, _ TBiSO `
~093/2114~ PCT/US93/03909
;~1333~S
29
Example 6 - Synthesis of Aliphatic_Subunits
3-EthoxY-5 5-dimethYlc~clohex-2-en-1-one_(7):
To a flame-dried 1 L round-bottomed flask containing
15.0 g (107 mmol~ 5,5-dimethylcyclohexane-1,3-dione and
120 mL absolute ethanol in 300 mL anhydrous benzene under
nitrogen atmosphere was added 750 mg p-toluenesulphonic
acid monohydrate (catalytic). The flask was fitted with
a Dean Stark trap for remo~al of water, and a ref~ux
condenser, and the mixture was heated to reflux for 9 h.
Upon cooling to room temperature, the solvent was removed
by rotary evaporation, and the residue was dissolved in
300 mL ethyl acetate. The organic solution was then
washed successively with 10~ aqueous NaOH (2 x 100 mL),
water ~1 x 100 mL), and brine (1 x 100 mL), dried over
Na2SO4, and concentrated under diminished pressure to give
16.5 g (92~) of the keto-enol ether as a pale yellow oil
(Rf 0.24, 2:1 hexanes/ethyl acetate) of greater than 98%
purity by lH NMR.
5.5-DimethylcYclohex-2-en-1-one (8): -
To a flame-dried 100 mL round-bottomed flask
containing lithium aluminum hydride (0.95 g, 2~.4 mmol, i~
O.5 mol equiv) in 35 mL anhydrous ether under nitrogen
atmosphere at 0C- was added 8.20 g (48.7 mmol) 3-ethoxy-
5,5-dimethylcyclohex-2-en-1-one (7) portionwise through a
syringe as a solution in 10 mL anhydrous ether. The
reaction mixture was aIlowed to warm to room temperature,
and after 4h, TLC analysis indicated complete consumption
of starting material. The reaction mixture was then
cooled to 0C before~the~cautious addition of 50 mL water,
and the contents of the flask were then poured into a 500
mL Erlenmeyer flask containing 150 m~ ice-cold 10~ H2SO4.
The mixture was the~ extracted with ether (2 x 200 mL),
and the combined organics were washed successively with
water (100 mL), and saturated aqueous NaHCO3 (100 mL),
dried over Na2SO4, and concentrated under diminished
pressure to give 6.05 g (quan~itative) of ~he
dimethylenone (Rf 0.5S, 2:1 hexane/ethyl acetate). l~ NMR
WOg3/2114~ PCT/US93/039~
;.~i33325 30
(400 MHz, CDCl3) ~ 1.05 (s, 6H, geminal-CH3~s), 2.23 (dd,
2H, CHCH2), 2.28 (s, 2H, COCH2), 6.03 (ddd, lH, 2-H), 6.87
ppm (ddd, lX, 3-H).
3-Ethoxy-6~5~5-trlmethyLc clohex-2-en-1-one (9):
To a flame-dried 300 mL round-bottomed flask
containing diisopropylamine (1.83 mL, 13.06 mmol, 1.1
equiv) in 50 mL anhydrous THF at -78C under nitrogen
atmosphere was added n-butyllithium (5.70 mL of a 2.2 M
solution in hexane, 12.50 mmol, 1.05 equiv). After 20 min
at -78C, 3-ethyoxy-5,5-dimethylcyclohex-2-en-1-one (7)
was added as a solution in 3 mL THF, and the reaction
mixture was allowed to stir at that temperature for 15 min
before gradual warming to 0C, and subsequent addition of -
iodomethane (3.5 mL, 59.5 mmol, 5 equiv). The reaction
15 mixture was then allowed to warm to room temperature, and i
after 3h, was quenched which saturated aqueous NH4Cl. The
contents of the flask were then extracted with 100 mL
ethyl acetate, and the organic phase was washed
successively with water (50 mL) and brine (50 mL), dried
over Na2SO4, and concentrated under diminished pressure.
Purification by flash column chromatography (silica gel,
hexane/ethyl acetate, gradient elution) afforded 1.94 g
(90%) of the methylated keto-enol ether (Rf 0.40, 2
hexanes/ethyl acetate) as a colorless oil.
4.~-5-Trime~hYlcyclohex-2-en-1-one (10): 1 -
- ~~ -This compound was prepared from 3-ethoxy-6,5,5- -
~ trimethylcyclohex-2-en-1-one (9) (1.60 g, 8.80 mmol) in
the manner previously described for enone 8, yielding 1.03
- g (85~) of the methylated enone as a colorless oil. lH NMR
- 30 (400 MHz, CDCl3) ~ 0.90 and 1.07 (2s, 2 x 3H, geminal-
. -CH3' s), 1 . 10 (d, 3H, CHCH3), 5 . 96 (dd, lH, 2-H), 6.16 ppm
~dd, lH, 3-H).
WO 93/21 145 ~ 1 3 3 3 2 S PCr/US93/03909
31
O O EtOHJpTsOH(cat) O OE~ 1- LAHJ Et2O
PhH / refhx J 9 h q~ 0 C ~ rt q~
J ~ J - -L J
92% ~ 2. lO'YoH2SO~ X
dimed~ 7 8
1. LDA ~ TE~
-78C
~ , 90% -
03~ o~
~ 10%~SO, ':
8~% 10
- . .
.. _
SUB5TITUTE SHEET
WO93/2114~ PCT/US93/~39~
2133325
32
Synthesis of CYclocvmo~Ql Analoqs
Example 7 '
6 - ~2 ' - (te~-ButYl ) dimethylsilvloxY-4~-bromo- 5~methoxy-
phenYll methyl-5 5-dimethvlcyclohex-2-en l-one (11):
To a flame-dried 50 mL round-bottomed flask
containing diisopropylamine (0.188 mL, 1.34 mmol, 1.1
equiv) in 10 mL anhydrous THF at -78C under nitrogen ~`atmosphere was added n-butyllithium (0.58 mL of a 2.2 M
solution in hexane, 1.28 mmol, 1.05 equiv). After 20 min
lo at -78C, 5,5-dimethylcyclohex-2-en-one (8) (0.151 g, 1.22
mmol) was added as a solution in 1 mL of THF, and the
reaction mixture was allowed to s~ir at that temperature
for 15 min, at which time the cooling bath was removed.
When the temperature of the reaction mixture (monitored
using a thermocouple probe) reached -5C, 2-(te~-
butyl)dimethylsilyloxy-4-bromo-5-methoxybenzylbromide (6)
(1.00 g, 2.44 mmol, 2.0 equiv) was added all at once as a
solution in 2 mL THF. The reaction mixture was then
allowed to warm to room temperature, and after 3 h, TLC
analysis indicated complete consumption of the enone
starting material, and the formation of a product of
intermediate polarity with respect to the two starting
components (Rf 0.59, 2:1 hexanes/ethyl acetate), and the
reaction was quenched by the addition of 5 mL saturated
aq~eous KHiC1. The contents of the flask were transferred
to a separatory funnel, and extracted with 60 mL ethyl
acetate, and the resultant organic phase was washed with
30i mL brine, dried over Na2SO4, and concentrated under
d-iminished pressure. Purification by flash column
.
chromatography (silica gel, hexane/ethyl acetate, gradient
--el~-ion) afforded 0.459 g (83%) of the benzylated enone as
-a- c~olorless, viscous oil, which solidified on standing.
2-r2'-(tert-Butyl~dimethYlsilYloxy-4'-bromo-5'-methoxy-
- ~hen~llmethY1-3.3-dimethYlcvclonexanone (12):
To a flame-dried 100 mL round-bottomed flask
containing 6-~2'-(tert-butyl)dimethylsilyloxy-4~-bromo-5'-
methoxyphenyl]methyl-5,5-dimethylcyclohex-2-en-1-one (11)
.
WO93/21l4~ 2 1 3 3 ~ 2 5 PCT/US93/03909
(0.154 g, 0.454 mmol) in 22 mL ethyl acetate (which had
been pre-dried over K2CO3) at room temperature was added 20
mg 5~ palladium on carbon, and after flushing/evacuating
the vessel 3 times with nitrogen, a hydrogen atmosphere
was introduced and maintained by use of a balloon. After
24 h, the flask was again flushed several times with
nitrogen, and the contents of the flask were filtered,
rinsing with an additional 100 mL ethyl acetate. Rotary
evaporation of the solvent afforded 0.15~ g ~quantitative)
of the saturated benzylic ketone as a colorless, viscous
oil (Rf 0.67, 2:1 hexane/ethyl acetate).
1-Methvlidene-2-(2'-hvdroxy-4'-bromo-5'-methoxy-
~henyl)methyl-3,3-dimethvlcyclohexane (racemic 1-
desbromocvclocymo~ol monomethyl ether) (13):
To a flame-dried 5Q mL round-bottomed flask
containing 2-[2'-(ten-butyl)dimethylsilyloxy-4~-bromo-5~-
methoxyphenyl]methyl-3,3-dimethylcyclohexanone (12) (0.130
g, O.28 mmol) in 5 mL anhydrous THF at -78C under
nitrogen atmosphere was added (trimethyl)silyme~hyllithium
(0.420 mL of a 1.0 M solution in pentane, 0.42 mmol, 1.50
equiv). An immediate change from colorless to a yellow
reaction solution was observed, and TLC analysis at that
time indicated complete consumption of starting material,
and the formation of a less polar product (Rf 0.79, 2
hexanes/ethyl acetate), and the reaction was subsequently
quenched with 4 mL saturated^aqueous NH4Cl. Ethyl acetate
.
(30 mL) extraction of the reaction- mixture, drying over
Na2SO4, and concentration under diminished press~re gave
0.152 g (quantitative) of a crude product, which appeared
to be a single diastereomer of the ketone addition product
by lH NMR analysis. A portion of this crude intermediate
(O.015 g, 0.028 mmol) was p~a-ced-in a 10 mL nalgene vial
containing 2 mL THF, 0.2 mL of a pre-made HF/pyridine
complex was added, and the mixture was allowed to stir at
room temperature for 32 h, at which time TLC analysis
indicated complete consumption of starting material, and
formation of a more polar product, having passed through
WO93/21145 PCT/US93/03~
~ ~333~ :
34
a most polar intermediate (confirmed as the desilylated
phenol by lH NMR). The contents of the reaction vessel
were transferred to a separatory funnel containing 20 mL
ethyl acetate and 10 mL 1.0 M NaHSO4. The layers were
separated, and the resultant organic phase was washed with
10 mL brine, dried over Na2SO4, and concentrated under
diminished pressure. Purificat'ion by flash column
chromatography (silica gel, hexanè'/ethyl acetate, gradient
elution) afforded 8.7 mg 192%) of the phenolic olefin as
a colorless oil. 1H NMR (400 MHz, CDCl3) ~ O.98 and 1.00
(2s, 2 x 3H, geminal-CH3's), 2.64 and 2.80 5d of ABq, 2H,
benzylic-CH2), 3.80 (s, 3H, OCH3) 4.36 and 4.63 (2s, 2 x
lH, olefinic-CH2), 6.58 and 6.95 ppm (2s, 2 x lH, Ar-H).
(This compound is also referred to as Compound '~C"
below.)
-- .
WO 93/2114~ 3 3 2 5 P~/US93/03909
o~1. LDA /1!~ 76 1 O C 8~ O~q
2. 2 e4u~v
~SO
6 83%
0~ . ~
H, / 5% Pd I C / E~OAC ~ q~
n 7~
O~.b ~S~ OAh
~ HFP~n~e ~
/ qu~tiv~ ~ ~>< 32h/92%
;:
1 ' ' ` ~ ' ' ' ''_ . ` ''
_. ~.
- ~
. . , ..
SUÇ3STITUTE SHEE~
~ ~ 3 3 2 ~ PCT/US93/039~
Example_8
1-Met hvl i dene-6- (2~ -a_cet oxy-4'-bro mo- 5~_
methoxY~hen~l)methyl-5 5-dimethy~lcyclohex-2-ene (14):
This compound was prepared from 6-[2~-(tert-
butyl)dimethylsilyloxy-4'-bromo-5'-methoxyphenyl]methyl-
5,5-dimethylcyclohex-2-en-1-one (11~ (0.075 g, 0.166 mmol)
in the manner previously described for olefin (13), with
the following procedural changes necessitated by the
incompatibility of structural fea~ures particular to this
substrate and the typical synthetic methodology. Upon
formation of the initial ~trimethyl)silylmethyllithium
addition adduct to enone 11, the phenolic protecting group
was exchanged prior to effecting elimination, using the
following protocol adhered to for all cyclocymopol analogs
possessing a 1,3-diene moiety as an extension of the
methylidene olefin. The crude addition product (0.090 g,
- 0.166 mmol) was dissolved in 5 mL anhydrous THF containing
0.20 mL acetic anhydride (large excess), and cooled to o~C
under nitrogen atmosphere. Tetra-n-butylammonium fluoride
(0.20 mL of a 1.0 M solution in THF, 0.20 mmol, 1.20
equiv) was added, and the mixture was allowed to warm to
room temperature. The contents of the flask were then
poured into a separatory funnel containing 30 mh ethyl
acetate and 10 mL 1.0 M NaHSO4, the layers were separated,
a~d the orga-nic phase was washed with 10 mL brine, dried
over Na2SO4-,-and concentrated under diminished pressure.
The crude material thus obtained was immediately carried
on to the next step by transferring to a 10 mL nalgene
via-l~containing 2-3 mL THF, and 0.3 mL premade HF/pyridine
complex was added. After stirring overnight at room
tempe~ature, the reaction mixture was worked up in the
usua~ manner, and purification by flash column
chromatography (silica gel, hexane/ethyl acetate, gradient
~elution) afforded 38.3 mg (64%) of the desired acetoxy-
diene as a colorless, oily solid. lX NMR (400 MHz, CDCl3)
~ 0.73 and 1.11 (2s, 2 x 3H, geminal-CH3's), 2.27 (s, 3H,
acetate-CH3), 3.83 (s, 3H, OCH3), 4.13 and 4.68 (2s, 2 x
~13332~
WO 93/~1 145 PCI /US93/03909 ~-
lH, C-CH2), 5 . 70 and 6 . 04 (2dd, 2 x lH, 2-H, 3-H), 6 . 52 and -~
7 . l9 ppm (2s , 2 x lH, Ar-H) .
(This compound is also designated Compound ~L~
below.)
Exam~le 9
l-MethYlidene-6-(2'-hydroxy-4'-bromo-5'-methoxvphenvl)
methyl-5 5-dimethvlcvclohex-2-ene (15):
In a lO m~ test tube was combined l-methylidene-6-
(2'-acetoxy-4'-bromo-5'-methoxyphenyl)methyl-5, 5 -
dimethylcyclohex-2-ene (14~ (10.0 mg, 0.026 mmol) and
2.0 mL of 5% methanolic K2CO3. After 10 min at room
temperature, the methanol was removed by ro;tary
evaporation, and the resultant residue was dissolved in
20 mL ethyl acetate. The organic solution was then washed
with saturated aqueous NH4Cl, dried over Na2SO4, and
concentrated under diminished pressure. Purification by
flash column chromatography (silica gel deactivated with ~;
triethylamine, hexane/ethyl acetate, gradient elution) ¦ `
afforded 7.1 mg (81~) of the phenolic diene as a
colorless, oily solid. ~H NMR (400 MHz, CDCl3) ~ O.73 and
1.16 (2s, 2 x 3H, geminal-CH3's), 3.82 (s, 3H, OCH3), 4.23
and 4.72 (2s, 2 x lH, C=CH2), 5.-78 and 6.08 ~2dd, 2 x lH, `
2-H, 3-H), 6.51 and 6.99 ppm (2s, 2 x lH, Ar-H).
(This compound is also designated Compound "K"
below.) ~ ~--
E-xam~le 10 j ` `
6- L2l-(tert-Bu~vl)dim~thvlsi-lyloxy-4l-bromo-sl-meth
phenvllmethYl-~ 5 5-trimethvl-cYclohe~-2-en-1-one (16):
This compound was p~epared from isophorone (0.168 g,
1.22 mmol) in the manner- previously described for
enone 11, affording 0.342 g (60%) of the alkylation
product (Rf 0.40, 2:1 hexane/ethyl acetate) as a
colorless, oily solid. I;~
,.
WO93/2114~ PCT/US93~39~
~ ~ 33~ S
38
.
1-Methylidene-6-(2'=acetoxy~-4'-bromo-5~-methoxY~henyl)
methYl-3,5,5-trimethylcyclohex-2-ene (17):
This compound was prepared from 6-[2'-(te~-butyl)
dimethylsilyloxy-4'-bromo-5'-methoxyphenyl]methyl-3,5,5-
trimethylcyclohex-2-en-1-one (16) (42.0 mg, 0.090 mmol) in
the manner previously described for acetoxy diene 14,
affording 18.4 mg (52~) of the acetoxy-diene as a
colorless oil. ~H NMR (400 MHz, CDCl3) ~ o.87 and 1.11
(2s, 2 x 3H, geminal-CH3's), 1.78 (s, 3H, olefinic-CH3),
2.26 (s, 3H, acetate-CH3), 3.83 (s, 3H, OCH3), 4.03 and
4.58 (2s, 2 x lH, C=CH2), 5.82 (s, lH, 2-H), 6.52 and 7.19
ppm (2s, 2 x lH, Ar-H). -
(This compound is also designated Compound "H"
below.)
15 Exam~le 1~ !
l-Methylidene-6-~2/-hydroxy-4r-bromo-s~-methoxy~hen
methyl-3 5 5-trimethylcYclohex-2-ene ~18):
This compound was prepared from 1-methylidene-6-(2'-
acetoxy-4'-bromo-5'-methoxyphenyl)methyl-3,5,5-
trimethylcyclohex-2-ene (17) (6.8 mg, 0.017 mmol) in the
manner previously described for phenolic diene 15,
affording 5.8 mg (96%? f the phenolic diene as a
colorless, oily solid. l~ NMR (400 MHz, CDCl3) ~ O.74 and
1.15 (~s-,~ ~ x 3H, geminal-CH3's), 1.79 (s, 3H, olefinic-
CH3), i.26 ~(s,~ 3H, acetate-CH3), 3.81 (s, 3H, OCH3), 4.16
and 4.62 (2s, 2 x lH, C=CH2), 5.87 (s, lH, 2-H), 6.52 and
61.99 ppm (2s, 2 x lH, Ar-H).
,. . - .
'13332S
WO 93/21145 - PCr/l lS93/03909
39
'- OM- :
~ BAP / THF / Ac2O / 0 C
3. HF/pyndine/T~ ~ .
14 ~:
0~ .:
CO3 / MeOH
~ L LDA/I~:/0C
60# ~ 32% rff:~v'd 5m
o~ 17
K~CO3/MeOH ~
: j ~o :
- .~
,,
.. .,
SVE~TITUTE SHEET
WO 93/21145 PCr/US93/039
21333'~5 :
Exam~le 12
trans-6- [2 ' - ttert-ButYl)dimethylsilyloxy-4'-bromo-5'-methoxY-
phenvll methyl-4 5,$-trlmethylcyclohex-2-en-1-one (19):
This compound was prepared from 4,5,5-trimethylcyclo-
5 hex-2-en-1-one (10) (169 mg, 1.22 mmol) in the manner
previously described for benzylated enone 11, affording
0.337 g (59g~) of the less polar trans diastereomer as a
colorless, oily solid, alo~g with 22 mg (4%) of the more
polar cis diastereomer as a colorless, oily solid,
10 separable by flash column chromatography. The relative
stereochemistry of each respective diastereomer was con-
firmed by nOe NMR experiments.
trans-2- r2 ~ - (tert-Butvl)dimeth~lsilYlo~-4~-bromo-5~-meth
phen~ll meth~rl-3.3.4-trimethylcyclohexanone (20):
This compound was prepared from trans-6-[2' -(tert-butyl)
dimethylsilyloxy-4'-bromo-S'-methoxyphenyl]methyl-4,5,5-
trimethylcyclohex-2-en-l-one (19) (150 mg, 0.321 mmol) in
the manner previously described for benzylated ketone 12,
affording 0.149 g (99%) of the trans ketone as a
20 colorless, oily solid.
trans -1-Methylidene- 6 - t2'-acetoxY-4'-bromo-5'-methox~n?henYl)
methyl-4,5.5-trimethYlcyclohex-2-ene (21):
- . -.
This~~compound was prepared from trans-6- [2' -(tert-butyl)
dimethylsilyloxy-4'-bromo-5'-methoxyphenyl]methyl-4,5,5-
25 trimethylcy~lohex-2-en-1-one (19) (70.0 mg, 0.15 mmol) in
the manner ~ previously described for acetoxy-diene 14,
affording 46.8 mg (79%) of the acetoxy-diene as a
colorless oil. ~lH N~ (400 MHz, CDCl3) ~ 0.76 and 1.14
(2s,--2 x 3H, geminal-CH3's), 1.00 (d, 3H, CHCH3), 2.30
(s, 3~I, acetate-CH3), 3.85 (s, 3H, OCH3), 4.12 and 4.67
(2s;-2--x-lH, C=CH2), 5.46 (d, lH, 2-H), 6.01 (dd, lH, 3-H),
6.53 and i.21 ppm (2s, 2 x lH, Ar-H).
(This compound is also designated Compound ~N~
below.)
~133325
~0 93/21 l45 PCr/US93103909
41
:
Exam~le 13
trans-1-MethYlidene-6 - 12 ' -hydroxy-4 ' -bromo-5 ' -methoxyphenYl)
methyl - 4 5, 5 - t rlmethyl c~clQhex- 2 - ene ( 2 2 ) :
This compound was prepared from trans-l-methylidene-6-
( 2 ' - acetoxy- 4 ' - bromo - 5 ' -methoxyphenyl ) methyl - 4, 5, 5 -
trimethylcyclohex-2-ene (21~ ~5 . 5 mg, 0 . 014 mmol) in the
manner previously described for phenolic diene 15,
affording 4.1 mg (84%) of the phenolic diene as a
colorless, oily solid. lX N~ (400 MHz, CDCl3) ~ o . 74 and
1.18 (2s, 2 x 3H, geminal~CH3's), 1.00 (d, 3H, CHCH3), 3.80
(s, 3H, OCH3), 4.22 and 4.70 (2s, 2 x lH, C=CH2), 5.52
(d, lH, 2-H), 6 . 04 (dd, lH, 3 -H), 6 . 50 and 6 . 99 ppm (2s , 2
x lH, Ar-H) .
I
WO 93/21 145 PCr/VS93/039~,
~ 1333'~S
i' 42
OM9
q~q 1. LDA / I~IF / 0 C ~dq ~ !
æ 6J~ *~
/ ~ 63%,. 15 / 1 ~ans: c TBSO
19 .,,
OMo .
H~/5%PdonC
EtQAc / 999'~ TBSO
.
1. Me~SiCH2Li / T~ / -78 C Br
0
- --- -: 'æ' TBAF J~ / Ac20
TBSO J~ 3. HF / pyridine / T~ 1' ,~
19 :Ohh 21
I~CO3~MeOH
84~ HO
22
~13332S
WO93/2114S PCT/US93/03909
' 43
Example_l4
trans -1-Methylidene-2-(2'-hydroxy 4'-bromo-5'-methoxyphenyl)
methYl-3 3 4-trlmethylcyclohexane (23):
This compound was prepared from trans-2- [2' - (te~-butyl)
dimethylsilyloxy-4'-bromo-5'-methoxyphenyl]methyl-3,3,4-
trimethylcyclohexanone ~20) (47.0 mg, 0.089 mmol) in the ~
manner previously described for phenolic olefin 13 to -'
afford 22.6 mg (72~) of the trans phenolic olefin as a -
colorless, oily solid. lH NMR (400 MHz, CDCl3) ~ O.79 and
10 1 . 08 (2s, 2 x 3H, geminal-CH3' s), 0 . 85 (d, 3H, CHCH3), 3 . 80
(s, 3H, OCH3), 4.36 and 4.58 (2s, 2 x lH, C=CH2), 6.50 and ~'
6.99 ppm (2s, 2 x lH, Ar-H). ~'
Exam~le 15 i~'
6-(4'-Nitrophenyl)methyl-S.S-dimethvlcYclohex-2-en-1-one
lS ~24)
This compound was prepared from 5,5-dimethylcyclohex-
2-en-1-one (8) (0.600 g, 4 . 83 mmol) and p-nitro~enzyl
bromide (1. 581 g, 7.32 mmol) in the manner described for
the synthesis of enone 11, affording 627 mg (50~) of the
20 nitro-enone as a pale yellow oil. ~X NMR (400 MHz, C~Cl3) ''
- ~ 1.02 and 1.18 (2s, 2 x 3H, geminal-CH3's), 2.83 and 3.10
(d of ABq, 2H, benzylic-H's), 6.0-0 (ddd,- lH, 2-H), 6.81
(ddd, lH, 3-H), 7.38 and 8.10 ppm (2d, 2 x 2H, Ar-H).
Example 16
1-Methvlidene-6-~3'-methyl-4'-nitrophenyl)methvl-5 5-
dimethylcvclohex-2-ene (25): :~
This compound was prepared from 6-(4'-nitrophenyl)
methyl-5,5-dimethylcyclohex-2-en-1-one (24) (133 mg, 0.514
mmol) in the manner pre~iously described for the synthesis
of olefin 13, with the following~' procedural changes.
Three equivalents of (trimethyl)silylmethyllithium were "
used, and the subsequent elimination step required 48 h to
go to completion, affording 120 mg (86%) of the nitro-
diene as a colorless oil. ~H NMR (400 MHz, CDC13) ~ O . 92
and 1.12 (2s, 2 x 3H, geminal-CH3's), 2.58 (s, 3H, Ar-CH3~, .
~,
. .
WO93/21145 PCT/US93/039~
~ 1 3 3
44
4.05 and 4.64 (2s, 2 x lH, methylidene-CH2), 5.70 (ddd, lH,
3-H), 6.03 (dd, lX, 2-H), 6.98 (s, lH, Ar-H), 6.99 and
7.88 ppm (2d, 2 x lH, Ar-H).
(This compound is also designated Compound "M"
below.)
Exam~le 17
1-MethYlidene-2-~2'-hydroxy-4'-bromo-5'-methoxyphenyl)
methylcyclohexane (26):
This ~ompound was prepared in three steps from
cyclohexanone and 2-(te~-butyl)dimethylsilyloxy-4-bromo-5-
methoxybenzyl bromide ~6) as previously described for the
synthesis of olefin 13, to give the desired olefin in
three steps in 13.5~ overall yield as a colorless oil.
1~ NM~ (400 MHz, CDCl3) ~ 2.54 and 2.92 (d of ABq, 2H,
benzylic-H's), 3.84 (s, 3H, OCH3), 4.46 (s, lH, OH), 4.62
and 4.71 (2s, 2 x lH, methylidene-CH2), 6.65 and 6.98 ppm
(2c, 2 x lH, Ar-H).
1,
'133325
,~'0 93/2114~ 45 P~/US93/03909 ~
~b ~ `.
~Q~ 1. Me3SiCH2~ / ~ / 78 C ~¢~
23 `'
~ "H"~
8 E~r 2~ 25
50% ' i:
o~O 1 LDA~ 0~
6 ~SO T~ / 23%, T8SO -
60% 26 1,
SUB~TITUl E SHEET
WO93/2114~ PCT/US93/039~
~ ~ 333`~
46
Semisynthetic Derivatives of Natural Cyclocvmopols
Example 18
(3R)-1-Debromocycloc ~ o~ol monomethyl ether (27L:
To a flame-dried 10 mL round-bottomed flask
containing (3~)-2'-(te~-butyl)dimethylsilyloxycyclocymopol
monomethyl ether (19.5 mg, 0.036 mmol) in 1 mL anhydrous
benzene with 1-2 mg AIBN at room temperature was added
n-Bu3SnH (39 ~L, 0.144 mmol, 4.0 equiv). After so min, TLC
analysis indicated virtually complete consumption of
starting material, and formation of a slightly less polar
product. Carbon tetrachloride ~200 ~L) was added, and
after 1 h at room temperature followed by 1.5 h at 0C,
2 mL THF and 200 ~L 1.0 M tetrabutylammonium fluoride --
- solution in THF were added. After 10 min at 0C, pH 7
buffer was added, and the reaction mixture was extracted
with hexane. The resultant organic solution was dri`ed
over Na2S04, and concentrated under diminished pressure.
Purification by flash column chromatography (silica gel,
lO~ ethyl acetate in hexane) afforded 7.5 mg (61%) of the
debromophenol as a colorless oil. The 400 MHz lH NMR
spectrum and TLC elution properties of this compound were ~d.- '
identical to those reported for the racemic analog 13.
--(This-- compound is also designated Compound "J"
below.)
Example i9 -- --
t3S)-l-Debromocyclocvmo~ol monomethyl ether (28):
I This compound was prepared from (3S) -2' - (te~-butyl)
dimethylsilyloxycyclocymopol monomethyl ether (25 . O mg,
O.Q47 mmol) in the manner described for the synthesis of
the-~ycIocymopol derivative 27, affording 5.5 mg (35%~ of
the- de~romophenol as a colorless oil, along with the
remainder of the mass balance as deprotected starting
material. The 400 MHz lH NMR spectrum and TLC elution
properties of this compound were identical to those
reported for the racemic analog 13.
NO93/21145 ~13 ~ 3 ~ 5 PCT/US93/03909
(This compound is also~ designated Compound ~G"
below.)
ExamPle 20
(3R)-4' -FormylcYclocymopol monomethyl ether (29):
To a flame-dried 10 mL round-bottomed flask
containing (3R)-2'-te~-butyl~dimethylsilyloxycyclocymopol
monomethyl ether (53.4 mgr 0.10 mmol) in 2 mL anhydrous
THF at -78C was added n-butyllithium (70 ~L of a 2.15 M
solution in hexane, 0.15 mmol, 1.50 equiv) all in one
portion. After 10 min at -78Cr N-methyl-N-~2-pyridyl)
formamide (28.5 mgr 25 ~Lr 0.21 mmol, 2.1 equiv) was added
as a solution in 1 mL THF, and the reaction mixture was
allowed to stir for 30 min before quenching with 1 mL 1:4
acetic acid/methanol. The reaction mixture was then
partitioned between hexane and 1.0 M NaHSO4r washed with
pH 7 bufferr and the resultant organic phase was dried
over NazSO4 and concentrated under diminished pressure.
Purification by radial chromatography (silica gelr 1 mm
chromatotron plater 10-15% ethyl acetate in hexane) gave
28.1 mg (58~) of the protected phenolic aldehyde as a
white solid. A portion of this silylated phenol (3.5 mg)
was deprotected using 25 ~L of 1-.0 M tetrabutylammonium
fluoride in 1 mL THF at 0C. ~~he reaction was quenched
with pH 7 buffer, and partitioned between hexanes and
water. The resultant organic phase was_dried o~er Na2SO4
and concentrated under diminished pressure. Purification
by radial chromatography (silica gel, 1 mm chromatotron
plate, 15~ ethyl acetate in hexanes) gave 2.1 mg ;79~) of
the desired aldehyde as a white sol-id. 1~ NMR (400 MHz,
CDCl3) ~ 1.10 and 1.26 (2s, 2 x 3~, geminal-CH3's), 2.70
and 3.01 (d of ABq, 2H, benzylic-~-s), 3.84 (s, 3H, OCH3),
4.39 and 4.62 ~2s, 2 x lH, methylidene-CH2), 4.44 (dd, lH,
BrCH), 6.60 and 7.27 (2s, 2 x lH, Ar-H), 10.33 ppm (s, lH,
C~O) .
WO93/21~ 3 3 3 2 ~ PCT/US93/039
48
ExamDle 21
(3R) -4' -Iodocyclocymo~ol monomethyl_ether t30):
This compound was prepared from (3R)-
2'-(te~-butyl)dimethylsilyloxycyclocymopolmonomethylether
(15.2 mg, 0.029 mmol) and iodine (300 ~L of a 0.25 M
solution in benzene, o.075 mmol, 2.6 equiv) in the manner
previously described for cyclocymopol derivative 29,
affording 9.2 mg (70~) of the iodocyclocymopol derivative
as a colorless oily solid. 1H NMR (400 MHz, CDC13) ~ 1.08
and 1.22 (2s, 2 x 3H, geminal-CH3's), 2.52 and 2.91 (d of
ABq, 2H, benzylic-H's), 3.78 (s, 3H, OCH3), 4.30 and 4.62
(2s, 2 x lH, methy1idene-CH2), 4.42 (dd, lHj ICH), 4.49
(s, lH, OH), 6.45 and 7.10 ppm (2s, 2 x lH, Ar-H).
(This compound is also designated Compound IIV'
below.)
Example 22
.
(3R~SR)-S-Hydroxyc~clocymopol monomethyl ether (31):
To a flame-dried 25 mL round-bottomed flask
containing (3R)-2'-(te~-butyl)dimethylsilyloxycyclocymopol
monomethyl ether (13.8 mg, 0.026 mmol) in 3.5 mh anhydrous
dichloromethane at room temperature was added selenium -
dioxide (l.S mg, 0.013 mmol, 0.50 equiv) and anhydrous t-
butyl hydroperoxide (17.3 ~L of a 3.0 M solution in 2,2,4-
trimetXy-lpentane, 0.052 mmol, 2.00 equiv), and the mixture
was allowed~to-stir at room temperature for 40 h, at which
time the solvent was removed by rotary evaporation.
Purification-by flash column chromatography (silica gel,
- hèxanes/ethyl acetate, gradient elution) afforded 13 mg of
material which was dissolved in 1 mh THF, cooled to 0C,
and treated with tetrabutylammonium fluoride (0.03 mL of
a 1.0M solution in THF, 0.030 mmol, l.lS equiv). After 10
min, the reaction was quenched by the addition of pH 7
buffer (5 mL~, and~extracted with hexanes (2 x 25 mL~.
The combined organic extracts were dried over Na2SO4, and
concentrated under diminished pressure. Purification by
W093J21145 ~1 3 3 3 2 5 PCT/US93/03909
4g
flash column chromatography (silica ~gel, hexanes/ethyl
acetate, gradient elution) afforded 8.8 mg ~78~) of the
hydroxycycloc~mopol as a white sold. lH NMR (400 MHz,
CDCl3) ~ 1.04 and 1.23 (2s, 2 x 3H, geminal-CH3's), 3.83 ~.
(s, 3H, OCH3), 4.42 (dd, lH, CHBr), 4.43 and 4.96 (2s,
2 x 2H, methylidene-CH2), 4.73 (dd, lH, CHOH), 6.54 and
7.00 ppm (2s, 2 x lH, Ar-H).
(This compound is also desi~nated Compound "E"
below.)
:
,
WO 93/21 14:~ PCl /US93~1)3~
`f~ 1 3332S
OMe OM~
Br~ l. n-Bu35nH / PhH ar~d~
~Br 2. ca4 ~ X
~SO 3. TBAF ~ T~ HO
- 61% 27
OMo
8r~¢~ ~ 1 n-3u35nH / PhH E~
TBSO 3. T~AP / ~7 HO
35%
- OMo O OMe
~ - 3. TBAP / T~
- ~ 7g% OM~
1. n-BuLi / ~ / r
11 I
2. I /b~z~e- ~ ~ ~ ~a-
3. TBAF/T~ I
79~o 3~ .
OM~ -- OMe OH
q~ 1. SeO2 / t-8uOOH ~r~ .
~X--8. 2. TBAF / ~E7 0
31
WO93/21145 ~1 3 ~ ~ 2 ~ PCT/US93/0~909
Representative derivative~compounds include:
o~ ~. .
Br~ Br~
, ~
O ~ ~ ~f
o
. .:
COMPOUND A COMPOUND B
1 -Methyl idene-2-~2'-acetoxy-4'-bromo-5 '- (3S)-1-Debromocyclocymopol monomethyl
methoxyphenyl)methyl-3,3-dimethylcyclohexane ether,2'-acetate
~ o~o~
o , '~
COMPC)UND C COMPOUND D
1-Methylidene-2-(2'-hydroxy-4'-bromo-5'- (3S)-1-Debromocyclocyms~pol monomethyl
methoxyphenyl)methyl-3,3~imethylcyclohexane ether,2'-methylcarbsnate
COMPOUND E COMPOU~ID F `
(3R,SR)-~Hydroxycyclocymopol monomethyl 2-(4'-Nitrophenyl)methylcyclohexanone10 ether
---
WO 93/21 145 PCr/US93/~39
2l333'~S 52
~r~
COMPOUND G COMPOU~D H
(3S)-1-Debromocyclocymopol monomethyl ether 1-Methylidene-6-(2'~acetoxy-4' bromo-5'-
methoxyphenyl)methyl-3 ,5,5-trimethylcyclohex-2-
ene
O~ ~ 3~
OH ~ ~ I .
COMPOUND I COMPOUND J
l-Methylidene-6-(4'-nitrophenyl)methyl-3,5,5- (3R)-1-Debromocyclocymopol monomethyl
trimethylcyclohex-2-ene ether ,.~.
' .
,, .
a~
-. . ~
COMPOUND K COMPOUND L
l-Methylidene-6-t2'~hyd~roxy-4'-bromo-5'- 1-Methylidene-6-t2'-acetoxy-4'-bromo-5'- . ¦ `~
methoxyphenyl)methyl-S,S~lmethylcyclohex-2-ene methoxyphenyl)methyl-5,5~imethylcyclohex-2-ene
WO 93/21145 i~ 3 3 ~ 2 ~ PCI~US9~3909
o~ ,
o*~
COMPOUND M COMPOUND N
,-Methylidene-6-(3'methyl4'-nitrophenyl)methyl- trans-1-Methylidene-6-(2'-acetoxy-4'-bromo-;'-
5,5-dimethylcyclohex-2-ene methoxyphenyl)methyl~,5,5-trimethylcyclohex-2-
ene
'~
l .
Q~b :
~ Off ~
COMPOUND O COMPOUND P ,
1-Methylidene-2-(4'-bromophenyl)methyl-3,3-1-Methylidene-2-~2'-hydroxy-4'-bramo-5'-
dlme~hylcyclohexane methoxyphenyl)methyl-3,3-dimethylcyclopentane
~ .
COMPOUND Q COMPOU~D R
1Methylidene-2~4'-nitrophenyl)methylcyclohexane(3R)-l-Debromocyclocymopol monomethyl .
ether,2'-methylcarbonate
WO 93/21145 PCl/US93/039~
') ~ 3332S
- 54
~ ~o ~;1~
COMPOUND S COMPOUND T
l-Methylidene-2-~2'-hydroxy-4'-bromo-5'- (3-R)-Cyclocymopol monomethyl ether,2'-
methoxyphenyl)methylcyclohexane methylcarbonate
~0~ ~
COMPOUND U COMPOUND V
(3R)-1-Debromocyclocymopol monomethyl (3R) 1'-lodocyclocymopol monomethyl ether
ether,2'-benzoate
-- .
. .
_. - - - .
,
~093/21145 ~1 3 3 3 2 5 PCT/US93/03909
Utilizing the "co-transfection~ assay~described
above, representative synthetic and semisynthetic
cyclocymopol derivatives have been tested and found to be
antagonists specifically for the intracellular receptor
for progesterone. Cultured monkey kidney cells (CV-l's)
were transfected with the human receptor cDNA for the
progesterone receptox altered at the Tau-1 locatlon and
utilized in the co-transfection assay. The assay was also
run using T47D (human breast cancer) cells. The
antagonist activity assay results are shown below in Table
1. Table 2 presents comparable results for the
antagonists RU-486 and (3R)-cyclocymopol monomethyl ether,
and for the agonist (3S)-cyclocymopol monomethyl ether.
Efficacy is reported as the ~ maximal response observed ;.
15 for each compound relative to RU-486, a compound known to j:
exhibit progesterone receptor antagonist activity. Also
reported in Tables 1 and 2 for each compound is its
potency or IC50 (which is the concentration (nM), required
to reduce the maximal response by 50~), and its binding
20 acti~ity for the progesterone receptor. .
.. .
Table 1 -
CV-l Cells T47D Cells ~
Com~ound Efficacy Potency Efficacy Potency Binding ..
t _ nM Kd,nM
A 55 225 70 200 ~ - 100-.
B 45 400 65 85 35~ -
c 55 400 70 200 85
D 45 450 55 150 50
E 85 450 85 475 925
F 100 550 80 200 925 ~,
G 45 600 50 300 -~ 2Q- ~:
H 40 700 60 775 60
I 95 700 75 125 55 .
J 95 750 8S 175 255
K 70 775 70 275 155
L 55 825 75 200 90
W O 93/21145 PCT~US93/039t
2~L3332S
56
CV-l Cells T47D Cells
Com~ound Ef f icacy Potency Ef f icacy Potency Binding
~ ~ ~ nM Kd, nM
M 90 875 80 350 75
N 90 1000 70 325 195
o 90 1000 85 850 445
P 100 1200 60 275 440
Q 95 1200 65 225 245
R 95 1225 85 250 230
S 90 . lS00 85 400 345
T 90 1575 90 300 185
U 9S 1600 80 300 lgO
V 95 1~00 85 325 95
Table 2
CV-l Cells T47D Cells
Ef f icacy Potency Efficacy Potency Binding
ComDound ~ . nM t nM Kd,nM
RU-486 100 0.1 - - 0.6
(3~)-cyclo- 85 965 85 385 450
: cymopol mono- i~
methy} ether ;
(3S) -cyclo-~20 ~lO,000 - - 325 . ~ ;
20 cymopol mono- - ,:~
methyl ether
The synthetic cyclocymopol compounds were also
individually tested for cross-reactivity with the other
known intracellular recèptor classes. This testing showed ::
the compounds not to have acti~ity with the glucocorticoid
receptor, ~in contrast to RU-486 which shows significant
activity for that receptor-. Some deri~ative compounds
were found to exhibit slight activity for the androgen
: receptor. - _.
- ...
30 Pharmacoloaical_and gther Applicatlons :;
It has been recognized that the co-transfection assay :
pro~ides a fùnctional assessment of the ligand being
tested as either an agonist or antagonist of the specific
genetic process sought to be affected, and mimics an ln
~VO 93/~1145 ,i~ ~ 3 3 3 2 5 PC~r/US93/03909
57
VlVO system in the laboratory. higands which do n~t react
with other intracellular receptors, as determined by the
co-transfection assay, can be expected to result in fewer
pharmacological side effects. Because the co-transfection
S assay is conducted in living cells, the evaluation of a
ligand provides an early indicator of the potential
toxicity of the candidate ligand at concentrations where
a therapeutic benefit would be expected.
As will be discernible to those skilled in the art,
the non-steroid progesterone receptor antagonist and
agonist compounds disclosed can be readily utilized in
pharmacological applications where progesterone receptor
antagonist or agonist activity is desired, and where it is
desired to minimize cross reactivities with other related
intracellular receptors. In v vo applications of the
invention include administration of the disclosed
compounds to mammalian subjects, and in particular to
humans.
The compounds of the present invention are small
20 ~molecules which are relatively fat soluble or lipophilic
and entér the cell by passive diffusion across the plasma
membrane. Consequently, these ligands are well suited for
admini~stration orally as well as by injection. Upon
administration, these~ ligands can selectively activate
progesterone receptors and thereby modulate processes
mediat~ed~by these receptors. - -`-
The pharmaceutical compositions of this invention areprepared in conventional dosage unit forms by incorpo-
:~ ! 1 rating an activé compound of the invention, or a mixture
; 30 of such compounds, with a nontoxic pharmaceutical ca-rrier
according to accepted procedures in a nontoxic amount
~;- sufficient to produce the desired pharmacodynamic a~ti~it~
in a mammalian and in particular a human subject. Prefer-~
ably, the composition contains the active ingredient in an
active, but nontoxic, amount selected from about 5 mg to
about 500 mg of active ingredient per dosage unit. This
-
WO93/2114~ PCT/~IS93/039~ -
213^332~ '
58
quantity~ depends on the-~ specific biological activity
desired and the condition of the patient.
The pharmaceutical carrier or vehicle employed may
be, for example, a solid or liquid. A variety of
pharmaceutical forms can be employed. Thus, when using a
solid carrier, the preparation can be plain mllled
micronized in oil, tableted, placed in a hard gelatin or
enteric-coated capsule in micronized powder or pellet
form, or in the form of a troche, lozenge, or suppository.
When using a liquid carrier, the preparation can be in the
form of a liquid, such as an ampule, or as an aqueous or
nonaqueous liquid suspension. The following examples
pro~ide illustrative pharmacological composition
formulations:
Exam~le 23
Hard gelatin capsules are prepared using the
followi~g ingredients:
Q u a n t i t y
~mq/caPsule)
(3R)-cyclocymopol monomethyl ether 140
Starch, dried 100
Magnesium stearate 10
Total - 250 mg
The above ingredients are mixed and filled into hard
gelatin capsules in 250 mg~q~a~titles.
Exam~le 24
A tablet is prepared using the ingredients below-
~~ Quantity
(mq/tablet )
(3R)-cyclocymopol mon-omethyl ether 140
Cellulose, microcrystalline 200
: Silicon dioxide, fumed 10
Stearic acid 10
Total 360 mg
.
h'O93/2114~ ~ 1 3 3 3 2 5 PCT/US93/03909
59
The components are blended and compressed to form tablets
each weighing 665 mg.
Example 25
Tablets, each containing 60 mg of active ingredient,
are made as follows:
Quantity
(mq/tablet)
(3R)-cyclocymopol monomethyl ether 60
Starch 45
Cellulose, microcry~talline 35
Polyvinylpyrrolidone (PVP)
(as 10~ solution in water) 4
Sodium carboxymethyl starch (SCMS) 4.5
Magnesium stearate 0.5
Talc 1.0
Total 150 mg
The active ingredient, starch, and cellulose are
passed through a No. 45 mesh U.S. sieve and mixed
thoroughly. The solution of PVP is mixed with the
resultant powders, which are then passed through a No. 14
mesh U.S. sieve. The granules so produced are dried at
- ~ 50C and pas-sed through a No. 18 mesh U.S. sieve. The
SCMS, magnesium stearate, and talc, previously passed
through a No. 60 mesh U.S. sieve, and then added to the
granules which, after mixing, are compressed on a ta~let~
machine to yield tablets each weighing 150 mg. ~ ~~
, , I
WO93/~1145 PCT/US93/039~ :
2~333'~S
Exam~le 26 --
Suppositories, each containing 225 mg of active
ingredient, may be made as follows:
(3R)-cyclocymopol monomethyl ether 225 mg
Saturated fatty acid glycerides 2 ! 040 mq :
Total 2,225 mg .
The active ingredient is passed through a No. 60 mesh U.S.
sieve and suspended in the saturated fatty acid glycerides
previously melted using the minimum heat necessary. The
mixture is then poured into a suppository mold of normal
2g capacity and allowed to cool. I
Exam~le.27 r, ~'
An intravenous formulation may be prepared as :
follows: .;
(3R)-cyclocymopol monQmethyl ether lO0 mg
Isotonic saline . l,000 ml `:
Glycerol lO0 ml ,l .
The compound`is dissolved in the glycerol and then I .`
the solution is slowly diluted with isotonic saline. The
20 solution of the above ingredients is then administered .
intravenously at a rate of l ml per minute to a patient. ` -:
:~ ~ . .
The compounds of this invention also have utility
when labeled as ligands for use in assays to determine the
presence of progesterone receptors-..-They are particularly
useful due to their ability t:o selectively activate
progesterone receptors, and can therefore be used to
determine the presence of such...recèptors in the presence
of other related receptors.
Due to the selective specificity of the compounds of
this invention for progesterone~ré~ceptors, these compounds
can be used to purify samples of progesterone receptors in
vi~o. Such purification can be carried out by mixing
samples containing progesterone receptors with one or more
of the cyclocymopol and derivative compounds disclosed so
that the compound (ligand) binds to the receptor, and then
~093/2114~ ~1 3 3 3 2 S PCT/US93/0390g -
separating out the bound ligand/receptor combinatio~ by
separation techniques which are known to those of skill in
the art. These techniques include column separation,
filtration, centrifugation, tagging and physical
separation, and antibody complexing, among others.
While the preferred embodiments have been described
and illustrated, various substitutions and modifications
may be made thereto without departing from the scope of
the invention. Accordingly, it is to be understood that
- 10 the present invention has been described by way of
illustration and not limitation.
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