Note: Descriptions are shown in the official language in which they were submitted.
CA 02371642 2001-10-29
WO 00/66592 PCT/LJS00/11836
-1-
CYCLIC UREA AND CYCLIC AMIDE DERIVATIVES
Field of the Invention
This invention relates to compounds which are agonists and antagonists of the
progesterone receptor, their preparation and utility.
Background of the Invention
Intracellular receptors (IR) form a class of structurally related gene
regulators
known as "ligand dependent transcription factors" (R. M. Evans, Science, 240,
889,
1988). The steroid receptor family is a subset of the IR family, including
progesterone
receptor (PR), estrogen receptor (ER), androgen receptor (AR), glucocorticoid
receptor (GR), and mineralocorticoid receptor (MR).
The natural hormone, or ligand, for the PR is the steroid progesterone, but
synthetic compounds, such as medroxyprogesterone acetate or levonorgestrel,
have
been made which also serve as ligands. Once a ligand is present in the fluid
surrounding a cell, it passes through the membrane via passive diffusion, and
binds to
the IR to create a receptor/ligand complex. This complex binds to specific
gene
promoters present in the cell's DNA. Once bound to the DNA the complex
modulates
the production of mRNA and protein encoded by that gene.
A compound that binds to an IR and mimics the action of the natural hormone
is termed an agonist, whilst a compound that inhibits the effect of the
hormone is an
antagonist.
PR agonists (natural and synthetic) are known to play an important role in the
health of women. PR agonists are used in birth control formulations, typically
in the
presence of an ER agonist: ER agonists are used to treat the symptoms of
menopause.
but have been associated with a proliferative effect on the uterus that can
lead to an
increased risk of uterine cancers. Co-administration of a PR agonist reduces
or ablates
that risk.
CA 02371642 2001-10-29
WO 00/66592 PCT/US00/11836
-2-
PR antagonists may also be used in contraception. In this context they may be
administered alone (Ulmann: et al, Ann. N Y. Acad. Sci. , 261. 248, 1995). in
combination with a PR agonist (Kekkonen, et al, Fertility and Sterility, 60,
610, 1993)
or in combination with a partial ER antagonist such as tamoxifen (WO 96/19997
Al.
July 4, 1996).
PR antagonists may also be useful for the treatment of hormone dependent
breast cancers (Horwitz; et al, Horm. Cancer, 283, pub: Birkhaeuser, Boston,
Mass.,
ed. Vedeckis) as well as uterine and ovarian cancers. PR antagonists may also
be
useful for the treatment of non-malignant chronic conditions such as fibroids
(Murphy.
et al, J. Clin . Endo. Metab. , 76, 513, 1993) and endometriosis (Kettel, et
al. Fertility
and Sterility, 56, 402, 1991).
PR antagonists may also be useful in hormone replacement therapy for post
menopausal patients in combination with a partial ER antagonist such as
tamoxifen
(US 5719136).
PR antagonists; such as mifepristone and onapristone, have been shown to be
effective in a model of hormone dependent prostate cancer, which may indicate
their
utility in the treatment of this condition in men (Michna, et al, Ann. N. Y.
Acad. Sci. ,
761, 224, 1995).
The compounds of this invention have been shown to act as competitive
inhibitors of progesterone binding to the PR and act as agonists and/or
antagonists in
fimctional models. either/or in-vitro and in-vivo. These compounds may be used
for
contraception, in the treatment of fibroids. endometriosis, breast, uterine,
ovarian and
prostate cancer, and post menopausal hormone replacement therapy.
Jones, et al, (U.S. Patent No. 5.688.810) describe the PR antagonist
dihydroquinoline 1.
CA 02371642 2001-10-29
WO 00/66592 PCT/US00/11836
-3-
N
Me
S
\ \
/ Me
H Me
1
Jones, et al, described the enol ether 2 (U.S. Patent No. 5,693,646) as a PR
ligand.
Me
Me
Me
Me
2
Jones, et al, described compound 3 (U.S. Patent No. 5,696,127) as a PR
ligand.
F
n
WO 00/66592 CA 02371642 2001-10-29
PCT/US00/11836
-4-
Zhi, et al, described lactones 4, 5 and 6 as PR antagonists (J. Med. Chem.,
41,
291. 1998).
0
\ O O Me
\ O Me I \ Me
/ / \ I / / \ / / \
~~ N Me '~ Me ~ ~Me
\ \ \ N O O /\N
H Me H Me H Me
4 5 6
Zhi. et al, described the ether 7 as a PR antagonist (J. Med. Chem., 41, 291.
1998).
\'
~O Me
/
Me
Me
l0
Combs, et al.: disclosed the amide 8 as a ligand for the PR (J Med. Chem., 38,
4880. 1995).
8
CA 02371642 2001-10-29
WO 00/66592 PCT/C1S00/11836
-5-
Penman, et. al., described the vitamin D analog 9 as a PR ligand (Tet.
Letters,
35. 2295. 1994).
CH3
9
Hamann, et al, described the PR antagonist 10 (Ann. N. Y. Acad. Sci., 761,
383,
1995).
OMe
1~
Chen, et al, described the PR antagonist 11 (Chen, et al, POI-37, 16"' Int.
Cong. Het. Chem., Montana, 1997).
WO 00/66592 CA 02371642 2001-10-29 pCT~S00/11836
-6-
11
Kurihari, et. al., described the PR ligand 12 (J. Antibiotics, 50, 360, 1997).
12
The patent by Christ et al. (WO 9814436) claims cyclo amide such as
compound A as inhibitors of HIV reverse transcriptase. Other prior art
includes
pyridazine cyclo amide such as compound B by Turck et al. (Tetrahedron. 49(3).
599-
606 (1993).) and compound such as C by Canonise et al. (.I. heterocyclic Chem.
26.
113(1989).). No activity data were reported in Turck's and Canonise'
publications.
WO X0/66592 CA 02371642 2001-10-29
PCT/US00/I 1836
_7_
N ~~
\~
F3C
CI / O /
\N~N~O N\N H O
H
A
w
N N O
H
C
Regarding cyclic amides, Singh et al. and Kumar et al. (Singh et al. J. Med.
Chem. 37(2), 248-254(1994); Kurnar et al. J. Org. Chem. 57(25), 6995-
6998(1992).)
disclose compounds such as D and E claimed as cAMP PDE III inhibitors.
D
Description of the invention
This invention provides compounds of Formula I:
WO 00/66592 CA 02371642 2001-10-29 pCT/US00/11836
-g_
Ri Rr
Ra
B
\D N Q
R3
wherein:
A. B and D are N or CH, with the proviso that A, B and D can not all be CH;
R' and RZ are independent substituents selected from H, C1 to C6 alkyl.
substituted C1 to C~ alkyl, Cz to C6 alkenyl, substituted CZ to C6 alkenyl, CZ
to C6
alkynyl, substituted CZ to C6 alkynyl, C3 to C8 cycloalkyl, substituted C3 to
Cx
cycloalkyl; aryl, substituted aryl, heterocyclic, substituted heterocyclic,
CORa,
NRBCOR~;
or R' and RZ are fused to form a spirocychc ring selected from a), b) or c),
each spirocyclic ring optionally substituted by from 1 to 3 C1-C3 alkyl
groups:
a) a 3 to 8 membered spirocyclic alkyl ring;
b) a 3 to 8 membered spirocychc alkenyl ring; or
c) an optionally substituted 3 to 8 membered heterocyclic ring
containing one to three heteroatoms from the group including O, S and N: the
spirocvclic rings of a), b) and c) being optionally substituted by from 1 to 4
groups
selected from fluorine, C1 to C~ alkyl, Cl to C6 alko~~-. C~ to C6 thioalkyl, -
CFA, -OH, -
CN, NHz, -NH(C1 to C6 alkyl), or -N(C1 to C6 alkyl)2;
RA is H, C1 to C3 alkyl. substituted C1 to C~ alkyl. aryl, substituted aryl,
C1 to
C3 alkok~~, substituted C1 to C~ alkoxy, C~ to C~ aminoallyl. or substituted
C1 to C
aminoalkvl:
RB is H, Cl to C3 alkyl, or substituted C~ to G alkyl;
R' is H. OH. NH2, C~ to C6 alkyl, substituted C~ to C6 alkyl, C3 to C6
alkenyl,
substituted C1 to C6 alkenyl, or CORD;
WO 00/66592 CA 02371642 2001-10-29 pCT/US00/11836
-9-
R~ is H, C1 to C3 alkyl, substituted C1 to C3 alkyl, aryl, substituted aryl,
C1 to
C3 alkoxy: substituted C1 to C3 alkoxy, C1 to C3 aminoalkyl, or substituted C1
to C;
aminoalkyl;
R4 is a trisubstituted benzene ring containing the substituents X, Y and Z as
shown below:
Y
~~~~Z
X-
X is taken from the group including halogen, CN, C1 to C3 alkyl, substituted
C,
to C3 alkyl, C1 to C3 alkoxy, substituted C1 to C3 alkoxy, C1 to C3
thioalkoxy,
substituted C1 to C3 thioalkoxy, amino, C1 to C3 aminoalkyl, substituted C1 to
C3
aminoalkyl, NO2, C1 to C3 pertluoroallcyl, 5 or 6 membered heterocyclic ring
containing 1 to 3 heteroatoms, CORD, OCORD, or NRECORD;
RD is H, C1 to C3 alkyl, substituted C1 to C3 alkyl, aryl, substituted aryl,
C1 to
C3 alkoxy, substituted C1 to C3 alkoxy, CI to C3 aminoalkyl, or substituted C1
to C3
aminoalkyl;
1~ RE is H, C1 to C~ alkyl, or substituted C~ to C3 alkyl;
Y and Z are independent substituents taken from the group including H.
halogen, CN, NOz, C~ to C~ alkoxy, C1 to C~ alkyl, or C~ to C3 thioalkoxy;
or
R4 is a five or six membered ring with 1, 2, or 3 heteroatoms from the group
including O, S, SO, SOZ or NRS and containing one or two independent
substituents
from the group including H, halogen, CN, N02 and C1 to C3 alkyl. C1 to C3
alkoxy, C~
to C; aminoalkyl, CORE, or NR~CORF;
RF is H, C~ to C3 alkyl, substituted CI to C3 alkyl, aryl, substituted aryl,
C~ to
C3 alkoxy; substituted C1 to C3 alkoxy, C1 to C3 aminoalkyl, or substituted C1
to C;
2~ aminoalkvl:
RG is H, C1 to C3 alkyl, or substituted C1 to C~ alkyl:
WO 00/66592 CA 02371642 2001-10-29
PCT/US00/11836
-10-
RS is H or C1 to C3 alkyl;
Q is O, S. NR6, or CR'Rg;
R6 is from the group including CN, C1 to C6 alkyl, substituted C1 to C6 alkyl,
C3 to C8 cycloalkyl, substituted C3 to Cg cycloalkyl, aryl, substituted aryl,
heterocyclic,
substituted heterocyclic, or SOZCF3;
R' and R8 are independent substituents from the group including H, C~ to C6
alkyl, substituted C, to C6 alkyl, C~ to Cg cycloalkyl, substituted C3 to Cg
cycloalkyl,
aryl. substituted aryl, heterocyclic, substituted heterocyclic, NO2, CN, or
COZR9;
R9 is C 1 to C3 alkyl:
or CR'R8 form a six membered ring of the structure below:
0
CH3
CH3
O
O
W is O or a chemical bond.
When W is a chemical bond, it is understood that Formula I exists as:
R~
Rz
R A
4
Q
Boo
R3
Preferred compounds are those of Formula I
WO 00/66592 CA 02371642 2001-10-29 pCT/US00/11836
-11-
R~ R2
4
R ~ A\
B~D N~Q
R3
I
wherein:
A. B and D are N or CH, with the proviso that A, B and D can not all
be CH.
Rl is H, CI to C6 alkyl, substituted C1 to C6 alkyl, C3 to C8 cycloallcyl,
substituted C3 to C8 cycloalkyl, aryl, substituted aryl, heterocyclic,
substituted
heterocyclic, CORA, or NRBCORA;
RZ is H, C1 to C6 alkyl, substituted C1 to C6 alkyl, CZ to C6 alkenyl,
substituted CZ to C6 alkenyl, C3 to C8 cycloalkyl, substituted C3 to Cg
cycloalkyl, aryl,
substituted aryl, heterocyclic, substituted heterocyclic, CORA, or NRBCORA;
or
R1 and R'' are fused to form the optionally substituted 3 to 8 membered
spirocvclic alkyl, alkenyl or heterocyclic rings described above;
RA is H, C1 to C3 alkyl, substituted C1 to C3 alkyl, ay l, substituted ar~~l,
C1 to
C3 alko~y, substituted C1 to C3 alkoxy, C1 to C3 aminoalkyl, or substituted C1
to C~
amino allyl;
RB is H, C1 to C3 alkyl, or substituted C~ to C~ alkyl;
R3 is H, OH; NHZ, C1 to C6 alkyl, substituted C~ to C6 alkyl, C3 to C6
alkenyl,
substituted C1 to C6 alkenyl, or CORD;
R~ is H, C~ to C4 alkyl, substituted C1 to C4 alkyl, awl. substituted aryl, C1
to
Ca alkoxy; substituted C1 to C4 alkoxy, C1 to Ca aminoalkyl, or substituted C1
to Ca
amino alkyl:
R4 is a trisubstituted benzene ring containing the substituents X. Y and Z as
shown below:
CA 02371642 2001-10-29
WO 00/66592 PCT/US00/11836
-12-
Y\ Z
X \\-
X is selected from halogen, CN, C1 to C3 alkyl, substituted C1 to C3 alkyl. C1
to C3 alkoxy, substituted C1 to C3 alkoxy, C1 to C3 thioalkoxy, substituted C1
to C3
thioalkoxy, C1 to C3 aminoalkyl, substituted C1 to C3 aminoalkyl, NOZ, C1 to C
perfluoroalkyl= a 5 membered heterocyclic ring containing 1 to 3 heteroatoms,
CORv,
OCORD. or NRECORD;
RD is H, C1 to C3 alkyl, substituted C1 to C3 alkyl, aryl, substituted aryl,
C1 to
C3 alkoy; substituted C1 to C3 alkoxy, C1 to C3 aminoalkvl, or substituted C1
to C
amino alkyl;
RE is H, C1 to C3 alkyl, or substituted Cl to C3 alkyl;
Y and Z are independent substituents selected from H, halogen, CN, NO2, C1
to C3 alkoxy, C1 to C3 alkyl, or C1 to C3 thioalkoxy;
or
RS is a five or six membered ring with l, 2, or 3 heteroatoms from the group
including O. S, SO, SOZ or NRS and containing one or two independent
substituents
from the group including H, halogen, CN; NOZ and C1 to C~ alkyl, or Cl to C~
alkoxv;
RS is H or C~ to C3 alkyl;
Q is O. S, NR~, or CR'R8;
R6 is selected from CN, C1 to C6 alkyl, substituted C1 to C6 alkyl, C~ to Cg
cycloalkyl, substituted C3 to Cg cycloalkyl, aryl. substituted aryl,
heterocyclic,
substituted heterocvclic, or SOzCF3;
R' and Rg are independent substituents selected from H, C1 to C6 alkyl,
substituted CI to C~ alk<~l. C; to C8 cycloalkyl, substituted C~ to C8
cycloalkyl, an~l.
substituted awl. heterocyclic. substituted heterocvclic, NO2, or CN COZR9;
R9 is C1 to Ca alkyl:
or CR'Rg form a six membered ring of the structure below:
WO 00/66592 CA 02371642 2001-10-29 PCT/LTS00/11836
-13-
0
CH3
\CH3
O
O
W is O or a chemical bond;
or a pharmaceutically acceptable salt thereof.
Still, more preferred compounds are those of Formula I
R~ R2
R4 A~ W
B~D N~Q
R3
I
wherein:
A, B and D are N or CH, with the proviso that A, B and D cannot all be CH;
R' = RZ and are selected from the group of C1 to C3 alkyl, substituted C1 to
C3
alkyl. or spirocyclic alkyl constructed by fusing R1 and RZ to form a 3 to 6
membered
spirocyclic ring;
R' is H, OH, NH2, C1 to C6 alkyl, substituted C1 to C6 alkyl, or CORD;
R~ is H, C1 to C4 alkyl, or C1 to C4 alkoxy;
R4 is a disubstituted benzene ring containing the substituents X, and Y as
shown below:
X
3'
4' ~
5' ~~
Y
WO 00/66592 CA 02371642 2001-10-29 PCT/US00/11836
-14-
X is selected from the group of halogen, CN, C~ to C3 alkoxy, C1 to C~ alkyl,
NOZ. C 1 to C3 perfluoroallcyl, 5 membered heterocyclic ring containing 1 to 3
heteroatoms, or C1 to C3 thioalkoxy;
Y is a substituent on the 4' or 5'position from the group including H,
halogen,
CN, NO2, C, to C3 alkoxy, C, to C4 alkyl, or C1 to C3 thioalkohy;
or
R4 is a five membered ring with the structure:
x'
Y'
U
U is O, S, or NRS;
R5 is H, or C1 to C3 alkyl, or Cl to C4 COZalkyl;
X' is selected from halogen, CN, NOZ, C1 to C3 alkyl, or Cl to C3 alkoxy;
Y' is selected from H and C1 to C4 alkyl;
or
R4 is a six membered ring with the structure:
1
IS
X1 is N or CX2;
XZ is halogen, CN or NO2;
Q is O. S. NR~, or CR~Rx
R6 is selected from CN, C1 to C6 all.~-1, substituted C~ to C6 alkyl. C~ to Cs
cvcloalkylj substituted C3 to C8 cycloalkyl, aryl, substituted aryl.
heterocyclic,
substituted heterocvclic. or SOZCF;;
CA 02371642 2001-10-29
WO 00/66592 PCT/US00/11836
-15-
R' and Rg are independent substituents selected from H, C1 to C6 alkyl,
substituted C1 to C6 alkyl, C3 to C8 cycloalkyl, substituted C3 to Cg
cycloalkyl, aryl,
substituted aryl, heterocyclic, substituted heterocyclic, NOZ, CN, or COzR9;
R9 is C1 to C3 alkyl;
or CR'Rg form a six membered ring of the structure below:
0
CH3
CH3
O
O
or a pharmaceutically acceptable salt thereof
Further preferred compounds include those of Formula I:
R~ R2
R4 A~ W
B~D N~Q
R3
I
wherein:
A, B and D are N or CH, with the proviso that A, B and D can not all be CH;
Rl = RZ and are selected from CH3 and spirocyclic alkyl constructed by fusing
R' and RZ to form a 6 membered spirocyclic ring;
R3 is H, OH, NHS, CH3, substituted methyl, or CORD;
R~ is H, CI to C; alkyl, or C1 to Ca alkoxy;
R4 is a disubstituted benzene ring containing the substituents X and Y as
shown
below:
CA 02371642 2001-10-29
WO 00/66592 PCT/US00/11836
-16-
X
3'
4, ~
5' ~~
Y
X is halogen, CN, methoxy, NO2, or 2-thiazole;
Y is a substituent on the 4' or 5'position selected from H and F;
or
R4 is a five membered ring of the structure:
x'
Y'
U
U is O, S, or NH;
X' is halogen, CN, or NO2;
Y' is H or C1 to Ca alkyl;
Q is O, S, NR6, or CR7Rg;
R6 is CN, C1 to C6 alkyl, substituted C1 to C6 alkyl, C3 to Cg cycloalkyl,
substituted C3 to Cg cycloalkyl, aryl, substituted aryl, heterocyclic,
substituted
heterocyclic, or SOZCF~;
R' and R8 are independent substituents selected from H, C1 to C6 alkyl,
substituted Ci to C6 alkyl, C3 to Cg cvcloalkyl, substituted C~ to Cg
cycloalkyl, ayl.
substituted aryl, heterocyclic, substituted heterocyclic, NOz, or CN COZR9;
R9 is CI to C~ alkyl;
or CR'R8 form a six membered ring of the structure:
0
0
~CH3
\~/ CHI
O
W~ 00/66592 CA 02371642 2001-10-29 PCT/LIS00/11836
-17-
W is O or a chemical bond;
or a pharmaceutically acceptable salt thereof.
Each of the generic and subgeneric groups of compounds described above. as
well as the methods of treatment and pharmaceutical compositions utilizing
them, may
be divided into two further subgeneric groups, one in which Q is oxygen and
another
in which Q is sulfur or NR6 or CR~Rg.
The compounds of this invention may contain an asymmetric carbon atom and
some of the compounds of this invention may contain one or more asymmetric
centers
and may thus give rise to optical isomers and diastereomers. While shown
without
respect to stereochemistry in Formula I, the present invention includes such
optical
isomers and diastereomers; as well as the racemic and resolved,
enantiomerically pure
R and S stereoisomers; as well as other mixtures of the R and S stereoisomers
and
pharmaceutically acceptable salts thereof.
The term "alkyl" is used herein to refer to both straight- and branched-chain
saturated aliphatic hydrocarbon groups having one to eight carbon atoms,
preferably
one to six carbon atoms; "alkenyl" is intended to include both straight- and
branched-
chain alkyl group with at least one carbon-carbon double bond and two to eight
carbon
atoms, preferably two to six carbon atoms; "alkynyl" group is intended to
cover both
straight- and branched-chain alkyl group with at least one carbon-carbon
triple bond
and two to eight carbon atoms, preferably two to six carbon atoms..
The terms "substituted alkyl", ''substituted alkenyl", and "substituted
alkynvl''
refer to alkyl. alkenyl, and alkynyl as just described having one or more
substituents
from the group including halogen, CN, OH, NOZ, amino, aryl, heterocyclic,
substituted
2~ aryl. substituted heterocyclic, alkoxy, aryloxy, substituted alkyloxy,
allcylcarbonyl,
alkylcarboxy, alkylamino, arylthio. These substituents may be attached to any
carbon
of alkyl, alkenyl, or alkynyl group provided that the attachment constitutes a
stable
chemical moiey.
WO 00/66592 CA 02371642 2001-10-29 PCT/US00/11836
-18-
The term ''aryl" is used herein to refer to an aromatic system which may be a
single ring or multiple aromatic rings fused or linked together as such that
at least one
part of the fused or linked rings forms the conjugated aromatic system. The
aryl
groups include but not limited to phenyl, naphthyl, biphenyl, anthryl,
tetrohydronaphthyl, phenanthryl.
The term "substituted aryl" refers to aryl as just defined having one to four
substituents from the group including halogen, CN, OH, NO2, amino, alkyl,
cycloalkyl,
alkenyl, alkynyl, alkoxy, aryloxy, substituted alkyloxy, alkylcarbonyl,
alkylcarboxy,
alkylamino, or arylthio.
The term "heterocyclic" is used herein to describe a stable 4- to 7-membered
monocyclic or a stable multicyclic heterocyclic ring which is saturated,
partially
unsaturated, or unsaturated, and which consists of carbon atoms and from one
to four
heteroatoms selected from the group including N, O, and S atoms. The N and S
atoms
may be oxidized. The heterocyclic ring also includes any multicyclic ring in
which any
of above defined heterocyclic rings is fused to an aryl ring. The heterocyclic
ring may
be attached at any heteroatom or carbon atom provided the resultant structure
is
chemically stable. Such heterocyclic groups include, for example,
tetrahydrofuran,
piperidinyl, piperazinyl, 2-oxopiperidinyl, azepinyl, pyrrolidinyl,
imidazolyl, pyridyl,
pyrazinyhpyrimidinyl, pyridazinyl, oxazolyl, isoxazolyl, morpholinyl, indolyl,
quinolinyl, thienyl, furyl, benzofuranyl, benzothienyl, thiamorpholinyl,
thiamorpholinyl
sulfoxide, and isoquinolinyl.
The term "substituted heterocyclic" is used herein to describe the
heterocyclic
just defined having one to four substituents selected from the group which
includes
halogen, CN; OH, NOZ, amino, alkyl, substituted alkyl, cycloallcyl, alkenyl,
substituted
2~ alkenyl, alkynyl, alkoxy, aryloxy, substituted alkyloxy, alkylcarbonyl,
alkylcarboxy,
alkylamino, or arylthio. The term "alkox~-" is used herein to refer to the OR
group_
where R is alkyl or substituted alkyl. The term ''aryloxy'' is used herein to
refer to the
OR group. where R is aryl or substituted awl. The term "alkylcarbonyl" is used
herein
to refer to the RCO group. where R is alkyl or substituted alkyl. The term
WO 00/66592 CA 02371642 2001-10-29
PCT/US00/11836
-19-
"alkylcarboxy" is used herein to refer to the COOR group, where R is alkyl or
substituted alkyl. The term ''aminoalkyl" refers to both secondary and
tertiary amines
wherein the alkyl or substituted alkyl groups containing one to eight carbon
atoms.
which may be either same or different and the point of attachment is on the
nitrogen
atom. "Halogen" refers to Cl, Br, F, or I.
The compounds of the present invention can be used in the form of salts
derived from pharmaceutically or physiologically acceptable acids or bases.
These
salts include, but are not limited to, the following salts with inorganic
acids such as
hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid and, as the
case may be.
such organic acids as acetic acid, oxalic acid, succinic acid, and malefic
acid. Other
salts include salts with alkali metals or alkaline earth metals, such as
sodium.
potassium, calcium or magnesium in the form of esters, carbamates and other
conventional "pro-drug" forms, which, when administered in such form, convert
to the
active moiety in vivo.
This invention includes pharmaceutical compositions and treatments which
comprise administering to a mammal a pharmaceutically effective amount of one
or
more compounds as described above wherein Q is oxygen as antagonists of the
progesterone receptor. The invention further provides comparable methods and
compositions which utilize one or more compounds herein wherein Q is S. NR6,
or
CR'R8 as agonists of the progesterone receptor.
The progesterone receptor antagonists of this invention, used alone or in
combination. can be utilized in methods of contraception and the treatment
and/or
prevention of benign and malignant neoplastic disease. Specific uses of the
compounds and pharmaceutical compositions of invention include the treatment
and/or
prevention of uterine myometrial fibroids, endometriosis, benign prostatic
hypertrophy:
carcinomas and adenocarcinomas .of the endometrium, ovaw, breast, colon.
prostate.
pituitary. meningioma and other hormone-dependent tumors. Additional uses of
the
present progesterone receptor antagonists include the synchronization of the
estrus in
livestock.
WO 00/66592 CA 02371642 2001-10-29
PCT/US00/11836
-20-
The progesterone receptor agonists of this invention, used alone or in
combination, can be utilized in methods of contraception and the treatment
and/or
prevention of dysfunctional bleeding, uterine leiomyomata, endometriosis;
polycystic
ovary syndrome, carcinomas and adenocarcinomas of the endometrium, ovary,
breast,
colon, prostate. Additional uses of the invention include stimulation of food
intake.
When the compounds are employed for the above utilities, they may be
combined with one or more pharmaceutically acceptable carriers or excipients,
for
example, solvents, diluents and the like, and may be administered orally in
such forms
as tablets, capsules, dispersible powders, granules, or suspensions
containing. for
example, from about 0.05 to 5% of suspending agent. syrups containing, for
example,
from about 10 to 50% of sugar, and elixirs containing, for example, from about
20 to
50% ethanol, and the like, or parenterally in the form of sterile injectable
solutions or
suspensions containing from about 0.05 to 5% suspending agent in an isotonic
medium Such pharmaceutical preparations may contain, for example, from about
25
to about 90% of the active ingredient in combination with the carrier, more
usually
between about 5% and 60% by weight.
The effective dosage of active ingredient employed may vary depending on the
particular compound employed, the mode of administration and the severity of
the
condition being treated. However, in general, satisfactory results are
obtained when
the compounds of the invention are administered at a daily dosage of from
about 0. 5 to
about 500 mg/kg of animal body weight, preferably given in divided doses two
to four
times a day. or in a sustained release form. For most large mammals, the total
daily
dosage is from about 1 to 100 mg, preferably from about 2 to 80 mg. Dosage
forms
suitable for internal use comprise from about 0.5 to 500 mg of the active
compound in
intimate admixture with a solid or liquid pharmaceutically acceptable carrier.
This
dosage regimen may be adjusted to provide the optimal therapeutic response.
For
example, several divided doses may be administered daily or the dose may be
proportionally reduced as indicated by the exigencies of the therapeutic
situation.
W~ 00/66592 CA 02371642 2001-10-29
PCT/C1S00/11836
-21 -
These active compounds may be administered orally as well as by intravenous,
intramuscular, or subcutaneous routes. Solid carriers include starch, lactose,
dicalcium
phosphate, microcrystalline cellulose, sucrose and kaolin, while liquid
carriers include
sterile water, polyethylene glycols, non-ionic surfactants and edible oils
such as com,
peanut and sesame oils, as are appropriate to the nature of the active
ingredient and the
particular form of administration desired. Adjuvents customarily employed in
the
preparation of pharmaceutical compositions may be advantageously included,
such as
flavoring agents, coloring agents, preserving agents, and antioxidants, for
example,
vitamin E, ascorbic acid, BHT and BHA.
The preferred pharmaceutical compositions from the standpoint of ease of
preparation and administration are solid compositions, particularly tablets
and hard-
filled or liquid-filled capsules. Oral administration of the compounds is
preferred.
These active compounds may also be administered parenterally or
intraperitoneally. Solutions or suspensions of these active compounds as a
free base or
pharmacologically acceptable salt can be prepared in water suitably mixed with
a
surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in
glycerol, liquid, polyethylene glycols and mixtures thereof in oils. Under
ordinary
conditions of storage and use, these preparations contain a preservative to
prevent the
growth of microorganisms.
The pharmaceutical forms suitable for injectable use include sterile aqueous
solutions or dispersions and sterile powders for the extemporaneous
preparation of
sterile injectable solutions or dispersions. In all cases, the form must be
sterile and
must be fluid to the extent that easy syringe abilin~ exits. It must be stable
under
conditions of manufacture and storage and must be preserved against the
2~ contaminating action of microorganisms such as bacterial and fungi. The
carrier can
be a solvent or dispersion medium containing, for example. water, ethanol
(e.g..
glycerol, propylene glycol and liquid polyethylene glycol), suitable mixtures
thereof.
and vegetable oil.
WO 00/66592 CA 02371642 2001-10-29 PCT/US00/11836
-22-
The compounds of this invention can be prepared following the Schemes
illustrated below:
As demonstrated in Scheme I, the compounds of this invention are generally
prepared by employing the suitable coupling reaction as a final step. An
appropriately
substituted ortho-amino acid or its derivatives such as ethyl ester (X = Br,
I, Cl, or a
latent coupling precursor such as alkoxy group which can be converted into OTf
group
suitable in the coupling reaction) is treated with a suitable organo metallic
reagent, e.g.
Grignard reagent. in appropriate nonprotic solvents which include but not
limited to
THF or ether to give ortho-amino carbinol 2 under an inert atmosphere such as
argon
or nitrogen at -78 °C to room temperature. Ring closure of carbinol 2
to yield oxazin-
2-ones 3 is commonly effected by a condensing agent such as
carbonyldiimidazole,
phosgene, dimethylcarbonate, or diethylcarbonate in a suitable nonprotic
solvent such
as THF at temperatures ranging from room temperature to 65 °C. The
arylation of
oxazin-2-ones 3 to yield 4 can be effected by various coupling reactions
including
Suzuki, Stille reactions etc.. These reactions are commonly performed in the
presence
of transition metallic catalyst, e.g., palladium or nickel complex often with
phosphino
ligands, e.g., Ph3P, 1,1'-bis(diphenylphosphino)ferrocene, 1,2-
bis(diphenylphosphino)ethane or palladium salt such as palladium acetate.
Under this
catalytic condition, an appropriately substituted nucleophilic reagent, e. g.
, ark l boronic
acid, arylstannane, or aryl zinc compound, is coupled with oxazinones 3 to
give
compounds 4. If a base is needed in the reaction, the commonly used bases
include but
not limited to sodium bicarbonate, sodium carbonate, potassium phosphate.
barium
carbonate, potassium acetate, or cesium fluoride. The most commonly used
solvents
in these reactions include benzene. DMF, isopropanol, ethanol. DME, ether.
acetone
or a mixture of any one of these solvent and water. The coupling reaction is
generally
executed under an inert atmosphere such as nitrogen or argon at temperatures
ranging
from room temperature to 95 °C. Oxazinones 3 can be converted into a
nucleophile
such as boronic acid which can be coupled with an appropriate electrophile,
e.g., ayl
WO 00/66592 CA 02371642 2001-10-29 pCT/US00/11836
-23-
bromide or aryl iodide, to yield 6 employing the coupling reaction condition
as
described above.
Scheme I
R R
COOR' OH
,A
RMgBr, TIC, ft, N2 \IT/'
B\D 'NH B\D NHz
z
1 2
R R
CDI, TEIF', 50 degrees C, N2'
D ~ O
3
R R
Ar
Arb(OI~2, Pd(Ph3P)4, Na2C03
DME/H20, N2, 85 degrees C '
~O
4
R R
n-BuLi, TI-IF', B(OMe)3 B~OH~z
-78 degrees C to rt, N2 '
B\D N "O
H
R 5
R
Ar
ArBr Pd(Ph3P)4, NaC03
DME/H20, 85 degrees C, N2
N O
H 6
R
Ar A
Lawesson's reagent
B\D N' S
H ~
The transformation of 3 into 5 can be effected by treating 3 with an organo
metallic
reagent; e.g., n-BuLi, in a nonprotic solvent such as THF or ether followed
b~~
quenching the reaction solution with a suitable electrophile such as trimethyl
borate.
triisopropyl borate, bishexalkyl tin reagent. or zinc chloride at temperatures
ranging
from-78 °C to room temperature under an inert atmosphere such as argon
or nitrogen.
CA 02371642 2001-10-29
WO 00/66592 PCT/US00/11836
-24-
Conversion of carbamate 6 to thiocarbamate 7 can be readily effected by
treatment of
6 with a suitable sulfur reagent such as PISS or Lawesson's reagent in a
suitable non-
protic solvent such as toluene, chlorobenzene, benzene, or xylene under an
inert
atmosphere such as argon or nitrogen at the temperature of boiling solvent.
Scheme II describes the procedures to prepare oxazinones bearing two
different substituents at position-4. The Weinreb amide 9 can be prepared from
an
appropriately substituted isatoic anhydride when treated with N-, O-
dimethylhydroxyl-
amine hydrochloride salt in a protic solvent such as ethanol or isopropanol at
reflux
under an inert atmosphere such as argon or nitrogen. Coupling of amide 9 with
an awl
electrophile such as aryl boronic acid or arylstannane to give 10 can be
effected by
employing a typical coupling reaction such as Suzuki, Stille coupling
procedure in a
similar fashion as described for the preparation of oxazinones 4. Treatment of
Weinreb amide 10 with organo metallic compounds, e.g., alkyllithium,
alkynyllithium,
aryllithium, or their Grignard counterpart in a nonprotic solvent such as THF
or ether
under an inert atmosphere such as argon or nitrogen at -78 ° to room
temperature
affords amino ketone 11. Conversion of ketone 11 to carbinol 12 can be
effected by
treatment of 10 with an organo metallic reagent such as alkyl, alkynyl, or ay
1 Grignard
compound in a nonprotic solvent such as THF or ether under an inert atmosphere
such
as argon or nitrogen at -78 °C to room temperature. Conversion of
ketone 11 to
carbinol 12 can also be effected by reduction of ketone group of 11 to the
caxbinol
moieWof 12 using an appropriate reducing reagent such as lithium aluminum
hydride.
sodium borohvdride in a suitable solvent such as THF, ether. or anhydrous
alcohol
under an inert atmosphere in the temperature ranging from 0 °C to the
boiling point of
the solvent. Ring closure of carbinol 12 to produce the compounds of this
invention.
13. can be accomplished with condensing agents such as carbonyldiimidazole,
phosgene, dimethylcarbonate; or diethylcarbonate in a suitable nonprotic
solvent such
as THF at temperatures ranging from room temperature to 65 °C.
Conversion of
carbamate 13 to thiocarbamate 14 can be readily effected by treatment of 13
with a
suitable sulfur reagent such as PISS or Lawesson's reagent in a suitable
nonprotic
WO 00/66592 CA 02371642 2001-10-29 pCT~,TS00/11836
-25-
solvent such as toluene, chlorobenzene, benzene, or xylene under an inert
atmosphere
such as argon or nitrogen at the temperature of boiling solvent.
Scheme II
O
X A X A
O EtOH, NHOMeMe, HCI, reflux
\ i
\
O ~ NHz
H o 9
ArB(OH)2, Pd(Ph3P~l, Na2C03 A
DME/H20, 85 degrees C, N2
NHz
R~i or R~VIgX, THF, -78 degrees C to rt'
11
O R
R~MgX, -78 degrees C to
rt, N2 _ A A
or reducing reagent
~
D NH2
12
R
CDI or triphosgene, THF R'
A A
0 degrees C to 65 degrees ~ \O
C '
H 13
R
R
,
Lawesson's reagent ~ A
A ~O
S
D
H 14
CA 02371642 2001-10-29
WO 00/66592 PCT/US00/11836
-26-
Alternatively, ortho-amino ketone 11 can be prepared by treatment of ortho-
amino nitrite 16 with an organo metallic compound such as organo lithium
reagent or
Gringard reagent in a suitable solvent such as THF' or ether under an inert
atmosphere
such as argon or nitrogen at temperatures ranging from -78 °C to room
temperature as
illustrated in Scheme III. Nitrite 16 can be readily prepared from an
appropriately
substituted nitrite such as bromobenzonitrile 15 using a suitable coupling
reaction such
as Stille or Suzuki protocol carried out in a similar fashion as described for
the
preparation of the Weinreb amide 10.
Scheme III
Ar
ArB(OH)2, Na2C03
g\ Pd(0), DME/H2O,N2
D ~ ~D
2
16
R6Li or R6MgX, 0 degrees C
Ar
w
TI h
11
The following non-limiting examples illustrate the compounds of the invention.
EXAMPLE 1
Z-Amino-5-bromo-3-pyridine carboxylic acid
To a solution of 2-amino-nicotinic acid (10 g; 72.5 mmol) in acetic acid (70
mL) was dropwise added bromine (9.8 mL. 79.8 mmol) at room temperature under
nitrogen. Upon completion of the reaction. the solvent was removed in vacuo,
the
WO 00/66592 CA 02371642 2001-10-29 PCT/US00/11836
-27-
residue triturated with ether and collected on a filter to give 2-amino-5-
bromo-3-
pyridine carboxylic acid as a yellow solid (15.7 g, 99%): mp 272 °C,
(decomposed);
1H-NMR (DMSO-db) 8 8.8-7.8 (bs, 2H), 8.44 (d, 1H, J = 2.48 Hz), 8.34 (d, 1H, J
=
2.48 Hz); MS (EI) m/z 216/218 ([M +]+, 100%)
EXAMPLE 2
2-(2-Amino-5-bromo-pyridin-3-yl)-propan-2-of
To a solution of 2-amino-S-bromo-3-pyridine carboxylic acid (5 g, 23 mmol) in
THF (70 mL) at 0 °C was added methyl magnesium bromide (13.7 g, 115
mmol) under
nitrogen. After addition, the reaction mixture was heated at 65 °C for
12 hours,
cooled to room temperature and quenched with saturated aqueous ammonium
chloride. The ethyl acetate was added and organic layer was separated, dried
over
sodium sulfate and concentrated. The residue was purified via flash
chromatography
to give 2-(2-amino-5-bromo-pyridin-3-yl)-propan-2-of as a yellow solid (1.2 g,
23%):
mp .107-108 °C; 1H-NMR (DMSO-d6) 8 7.89 (d, 1H, J= 2.3 Hz), 7.40 (d,
1H, J= 2.3
Hz), 6.28 (bs, 2H), 5.51 (s, 1H), 1.4 (s, 6H); MS (APCI) m/z 231 ([M + H]+,
100%).
EXAMPLE 3
6-Bromo-4,4'-dimethyl-1,4-dihydro-3-oxa-1,8-diaza-naphthalen-2-one
A mixture of 2-(2-amino-5-bromo-pyridin-3-yl)-propan-2-of (0.86 g, 3.7
mmol) and 1,1'-carbonyldiimidazole (excess) in THF (10 mL) was heated at 35
°C
overnight. The reaction solution was cooled to room temperature, poured into
ethyl
acetate (200 mL), washed with 1 N HCl (2x50 mL), dried over sodium sulfate,
and
concentrated. The residue was purified by a flash chromatography (silica gel,
25%
2~ eth~~l acetate/hexane) to afford 6-bromo-4,4'-dimethyl-1,4-dihvdro-3-oxa-
1,8-diaza-
naphthalen-2-one (0.9 g, 94%) as a white solid: mp 251-252 °C; 1H-NMR
(DMSO-
db) 8 10.9 (s, 1H), 8.32 (d, 1H; J = 2.19 Hz); 8.0 (d, 1H, J = 2.22 Hz), 1.6
(s, 6H);
MS (EI) miz 256 ([M]~. 80%); Anal. Calc. For C9H9BrNz02: C. 42.05, H. 3.~3, N,
10.90. Found: C. 42.15, H, 3.65, N. 10.80.
CA 02371642 2001-10-29
WO 00/66592 PCT/US00/11836
-28-
EXAMPLE 4
6-(3-Chloro-phenyl)-4,4-dimethyl-1,4-dihvdro-3-oxa-1,8-diaza-naphthalene-2-
one
A mixture of 6-bromo-4,4'-dimethyl-1,4-dihydro-3-oxa-1,8-diaza-naphthalen-
2-one (0.1 g, 0.39 mmol), 3-chlorophenyl boronic acid (0.075 g, 0.47 mmol),
tetrakis(triphenylphosphine)-palladium (0) (0.023 g, 0.02 mmol), and sodium
carbonate (0.1 g, 0.94 mmol) in DME (8 mL) and water (5 mL) was subject to a
blanket of nitrogen for 15 minutes at 50 °C and then was heated at 85
°C for 30
minutes. The reaction was cooled to room temperature and ethyl acetate (100
mL)
was added. The organic layer washed with aqueous ammonium chloride (2x50 mL)
and with brine (50 mL), dried over magnesium sulfate and concentrated. The
product
was dissolved in dichloromethane and passed through a plug of magnesol. The
solvent
was removed and the clear oil obtained triturated with ether (25 mL) to give 6-
(3-
chloro-phenyl)-4,4-dimethyl-1,4-dihydro-3-oxa-1,8-diaza-naphthalene-2-one as a
white solid (0.087 g, 80%): mp 214-215 °C; 1H-NMR (DMSO-d6) b 10.9 (s,
1H),
8.56 (d, 1H, J = 2.35 Hz), 8.06 (d, 1H, J = 2.35 Hz), 7.86 (t, 1H, J = 2.35
Hz), 7.72
(td, 1H, J = 9.4, 2.35 Hz), 7.54-7.44 (m, 2H), 1.69 (s, 6H); MS (ESI) mlz 289
([M+H]+, 100%); Anal. Calc. For C1~H1~N03: C, 62.40, H. 4.54, N, 9.70. Found:
C, 60.53. H, 4.40, N. 9.21.
EXAMPLE 5
6-Chloro-3-nitro-pyridine-2-carbonitrile
A mixture of 2. 6-dichloro-3-nitropyridine and cuprous cyanide in 1-methyl-2-
pyrrolidinone was quickly heated to 180 °C and maintained at that
temperature for 15
minutes with vigorous stirring. The mixture was cooled to -10 °C and
the deep brown
solution was poured into ice water ( 3.5 L) and stirred for 30 min. The
flocculent
brown precipitate was collected and washed with H20. After drying for about
1.5 h.
the moist solid was extracted with boiling toluene (3x300 mL). The combined
toluene
WO 00/66592 CA 02371642 2001-10-29 pCT/t,1S00/11836
-29-
extracts were washed with H20, brine, and dried (MgS04), concentrated. The
crude
product was crystallized from EtOAc/hexane to afford the title compound: mp
115-
117 °C; 1H NMR( DMSO-d6) 8 8.16 (dd, 1H, J = 8.7, 3.0 Hz), 8.82 (d, 1H,
J = 9.0
Hz)MS (EI) m/z 183/185 (M+H)+; Anal. Calc. For C6H2C1N302: C, 39.26, H,
1.10, N, 22.89. Found: C, 39.47, H, 1.38, N, 22.77.
EXAMPLE 6
3-Amino-6-chloro-nvridine-2-carbonitrile
To a solution of 2,6-dichloro-3-nitropyridine(4.8 g, 26.15 mmol) in MeOH (60
mL) and concentrated HCl (25 mL) was slowly added iron powder (5.12g, 91.53
mmol). After the completion of addition, the mixture was reffuxed for 45
minutes and
poured into 700 mL of HZO. Filtration of the resulting slurry gave a dull
yellow solid.
The filtrate was made basic with concentrated NH40H, the resulting slurry was
filtered and both the solid and the filtrates were extracted with ether. The
combined
extracts were dried (MgS04) and evaporated to give the title compound as a
creamy
solid (2.8g, 58%): mp 162-165 °C which was used in next step without
further
purification.
EXAMPLE 7
1-(3-Amino-6-chloro-nyridin-2-yl)-ethanone
To a solution of 3-amino- 6-chloro-pyridine-2-carbonitrile (0.75g, 4.88 mmol)
in anhydrous THF (25 mL) under nitrogen was added a solution of
methylmagnesium
bromide (3M in ether, 8.1 mL, 24.3 mmol). The reaction mixture was stirred for
30
minutes and then slowly quenched with H20, and treated with 5N HCl solution.
The
mixture was extracted with ethyl acetate (3x100 mL) and organic extracts were
washed with brine, and dried (MgS04). After removal of the solvent, the
residue was
purified by a chromatography using EtOAc/hexane mixture (1:3) as eluent to
afford
the title compound as a greenish crystalline solid: (0.71g, 85%): mp: 108-110
°C. 1H
WO 00/66592 CA 02371642 2001-10-29 PCT/US00/11836
-30-
NMR (DMSO-d6) 8 2.51 (s, 3H), 7.28-7.39 ( m, 4H); MS(ESI) m/z 171/173
(M+H)'; Anal. Calc. For C7H7C1N20: C, 49.28, H. 4.14, N. 16.14. Found: C.
49.70, H, 4.03, N. 16.41.
EXAMPLE 8
1-(3-Amino-6-chloro-pyridin-2-yl)-propan-2-of
To a solution of 1-(3-amino-6-chloro-pyridin-2-yl)-ethanone in anhydrous
THF under N2 was added a solution of methylmagnesium bromide (3N in ether).
The
resulting reaction mixture was stirred at room temperature for 4 h, then
slowly
quenched with H20, treated with 0.5 N HCl and stirred for 30 minutes, diluted
with
ethyl acetate, washed with brine, dried (MgS04), concentrated, and
chromatographed
using a mixture of EtOAc/Hexane ( 3.5: 6.5) to afford the title compound as a
white
crystals (0.45g; 82%): mp: 118-121 °C. 1H NMR(DMSO-d6) b 1.45(s, 6H),
3.35(s,
1H), 5.51(s, 1H), 5.68(s, 1H), 7.02(s, 1H); MS((+)APCI) m/z 187/189 (M+H)+;
Anal. Calc. For C8H11C1N20: C, 51.48, H, 5.94, N, 15.01. Found: C, 51.22, H,
5.99,
N, 14.47.
EXAMPLE 9
6 Chloro-4,4-dimethyl-1,4-dihvdro-3-oxa-1,5-diaza-naphthalen-2-one
To a solution of 1-(3-amino-6-chloro-3-nitro-pvridin-2-yl)-propan-2-of (0.3g,
1.67 mmol) in anhydrous THF (20 mL) was added a solution of 1,1'-
carbonvldiimidazole (0.688, 4.12 mmol) in anhydrous THF (10 mL). The reaction
mixture was heated under reflux for 3 h. The reaction mixture was treated with
O.SN
HCIwashed with brine. H20, dried (M8S04), concentrated and crystallized from
2> EtOAc/hexane to obtain the title compound as a white crystalline solid
(0.2g. 56%):
mp 175-178 °C. 1H NMR( DMSO-d6) d 1.60 (s, 6H), 7.30 (d, 1H, J= 8.41
Hz), 7.41
WO 00/66592 CA 02371642 2001-10-29 PCT/CTS00/11836
-31 -
(d, 1H, J = 8.41 Hz), 10.47 (s, 1H); MS((+)APCI) mlz 213 (M+H)+; Anal. Calc.
For
C9H9C1N202C, 50.84, H, 4.27, N, 13.17. Found: C. 50.99, H, 4.28, N. 12.98.
EXAMPLE 10
The compounds of this invention were tested in the relevant assay as described
below and their potency are in the range of 0.01 nM to 5 ~M in the in vitro
assays and
0.001 to 300 mg/kg in the in vivo assays. The selected example is example 4.
Me Me
N~ ~N~ ~O
H
Example 4, hPR CV-1, ICSO= 0.8 EtM
A. In-vitro biology
The in-vitro biology is determined by (1) competitive
Radioligand Binding: using the A-form of the human progesterone receptor with
progesterone as the radioligand; (2) co-transfection assay, which provides
functional
activity expressed as agonist EC50 and Antagonist IC50 values; (3) a T47D cell
proliferation, which is a further functional assay which also provides agonist
and
antagonist data; and (4) T47D cell alkaline phosphatase assay. which is a
further
functional assay which also provides agonist and antagonist data.
1. hPR Binding assay - This assay is carried out in
accordance with: Pathirana, C.; Stein, R.B.; Berger, T.S.; Fenical, W.;
Ianiro, T.; Mais,
D.E.; Torres, A.; Glodman, M.E., Nonsteroidal human progesterone receptor
modulators from the marine alga cymoplia barbata, J. Steroid Biochem. Mol.
Biol.,
1992, 41. 733-738.
CA 02371642 2001-10-29
WO 00/66592 PCT/US00/11836
-32-
2. PRE-luciferase assay in CV-1 cells
The object of this assay is to determine a compound's
progestational or antiprogestational potency based on its effect on PRE-
luciferase
reporter activity in CV-1 cells co-transfected with human PR and PRE-
luciferase
plasmids. The materials methods used in the assay are as follows.
a. Medium: The growth medium was as follows:
DMEM (BioWhittaker) containing 10% (v/v) fetal bovine serum (heat
inactivated). 0.1
mM MEM non-essential amino acids, 100U/ml penicillin, 100mg/ml streptomycin,
and
2 mM GlutaMax (GIBCO, BRL). The experimental medium was as follows:
DMEM (BioWhittaker), phenol red-free, containing 10% (v/v) charcoal-stripped
fetal
bovine serum (heat-inactivated), 0.1 mM MEM non-essential amino acids, 100U/ml
penicillin, 100mg/ml streptomycin, and 2 mM GlutaMax (GIBCO, BRL).
b. Cell culture transfection. treatment. and luciferase
assay
Stock CV-1 cells are maintained in growth medium.
Co-transfection is done using 1.2x10' cells, 5 mg pLEM plasmid with hPR-B
inserted
at Sphl and BamHl sites, 10 mg pGL3 plasmid with two PREs upstream of the
luciferase sequence, and 50 mg sonicated calf thymus DNA as carrier DNA in 250
ml.
Electroporation is carried out at 260 V and 1,000 mF in a Biorad Gene Pulser
II.
After electroporation, cells are resuspended in growth medium and plated in 96-
well
plate at 40,000 cells/well in 200 pl. Following overnight incubation, the
medium is
changed to experimental medium. Cells are then treated with reference or test
compounds in experimental medium. Compounds are tested for antiprogestational
activity in the presence of 3 nM progesterone. Twenty-four hr. after
treatment, the
medium is discarded. cells are washed three times with D-PBS (GIBCO, BRL).
Fifty
ul of cell lysis buffer (Promega Madison, WI) is added to each well and the
plates are
shaken for 15 min in a Titer Plate Shaker (Lab Line Instrument. Inc.).
Luciferase
activit<~ is measured using luciferase reagents from Promega.
WO 00/66592 cA 02371642 2001-l0-29 pC'I'/[TS00/11836
-33-
c. Ana~sis of Results:
Each treatment consists of at least 4 replicates. Log
transformed data are used for analysis of variance and nonlinear dose response
curve
fitting for both agonist and antagonist modes. Huber weighting is used to
downweight
the effects of outhers. ECSO or ICSO values are calculated from the
retransformed
values. JMP software (SAS Institute, Inc.) is used for both one-way analysis
of
variance and non-linear response analyses.
d. Reference Compounds:
Progesterone and trimegestone are reference progestins and
RU486 is the reference antiprogestin. All reference compounds are run in full
dose-
response curves and the ECSO or ICSO values are calculated.
Table 1. Estimated ECSp, standard error (SE),
and 95% confidence intervals
(CI) for reference progestins from three
individual studies 95%
EC50 CI
Compound Exp (nM) SE lower upper
Progesterone 1 0.616 0.026 0.509 0.746
2 0.402 0.019 0.323 0.501
3 0.486 0.028 0.371 0.637
Trimegestone 1 0.0075 0.0002 0.0066 0.0085
2 0.0081 0.0003 0.0070 0.0094
3 0.0067 0.0003 0.0055 0.0082
Table 2. Estimated
ICso, standard error
(SE), and 95% confident
interval (CI)
for the antiprogestin, from three idual studies
RU486 indiv
IC 50 95 % CI
Compound Exp (nM) SE lower upper
RU486 1 0.028 0.002 0.019 0.042
2 0.037 0.002 0.029 0.048
3 0.019 0.001 0.013 0.027
WO 00/66592 CA 02371642 2001-l0-29 PCT/US00/11836
-34-
Progestational activity: Compounds that increase PRE-luciferase activit5-
significantly (p<0.05) compared to vehicle control are considered active.
Antiprogestational activity: Compounds that decrease 3 nM progesterone
induced PRE-luciferase activity significantly (p<0.05)
ECSO: Concentration of a compound that gives half maximal increase PRE-
luciferase activity (default-nM) with SE.
ICSO: Concentration of a compound that gives half maximal decrease in 3 nM
progesterone induced PRE-luciferase activity (default-nM) with SE.
3. T47D cell proliferation assay
The objective of this assay is the determination of
progestational and antiprogestational potency by using a cell proliferation
assay in
T47D cells. A compound's effect on DNA synthesis in T47D cells is measured.
The
materials and methods used in this assay are as follows.
a. Growth medium: DMEM: F 12 ( 1:1 )
(GIBCO, BRL) supplemented with 10% (v/v) fetal bovine serum (not heat-
inactivated), 100U/ml penicillin, 100mg/ml streptomycin, and 2 mM GlutaMax
(GIBCO, BRL).
b. Treatment medium Minimum Essential
Medium (MEM) (#51200-038GIBC0, BRL) phenol red-free supplemented with 0.5%
charcoal stripped fetal bovine serum. 100U/ml penicillin, 200 mg/ml
streptomycin, and
2 mM GlutaMax (GIBCO, BRL).
c. Cell culture
Stock T47 D cells are maintained in growth medium. For BrdU incorporation
assay. cells are plated in 96-well plates (Falcon. Becton Dickinson Labware)
at 10,000
cells/well in growth medium After overnight incubation, the medium is changed
to
treatment medium and cells are cultured for an additional 24 hr before
treatment.
Stock compounds are dissolved in appropriate vehicle (100% ethanol or 50%
ethanol/50% DMSO), subsequently diluted in treatment medium and added to the
cells. Progestin and antiprogestin reference compounds are run in full dose-
response
CA 02371642 2001-10-29
WO 00/66592 PCT/US00/11836
-35-
curves. The final concentration of vehicle is 0.1 %. In control wells, cells
receive
vehicle only. Antiprogestins are tested in the presence of 0.03 nM
trimegestone, the
reference progestin agonist. Twenty-four hours after treatment, the medium is
discarded and cells are labeled with 10 mM BrdU (Amersham Life Science,
Arlington
Heights. IL) in treatment medium for 4 hr.
d. Cell Proliferation Assay
At the end of BrdU labeling, the medium is removed and BrdU
incorporation is measured using a cell proliferation ELISA kit (#RPN 250.
Amersham
Life Science) according to manufacturer's instructions. Briefly, cells are
fixed in an
ethanol containing fixative for 30 min, followed by incubation in a blocking
buffer for
30 min to reduce background. Peroxidase-labeled anti-BrdU antibody is added to
the
wells and incubated for 60 min. The cells are rinsed three times with PBS and
incubated with 3,3'5,5'-tetramethylbenzidine (TMB) substrate for 10-20 min
depending upon the potency of tested compounds. Then 25 pl of 1 M sulfuric
acid is
1 S added to each well to stop color reaction and optical density is read in a
plate reader at
450 nm within 5 min.
e. Ana~sis of Results:
Square root-transformed data are used for analysis of variance and nonlinear
dose response curve fitting for both agonist and antagonist modes. Huber
weighting is
used to downweight the effects of outliers. ECSO or ICso values are calculated
from
the retransformed values. JMP software (SAS Institute, Inc.) is used for both
one-way
analysis of variance and non-linear dose response analyses in both single dose
and dose
response studies.
f Reference Compounds:
Trimegestone and medroxyprogesterone acetate (MPA) are reference
progestins and RU486 is the reference antiprogestin. All reference compounds
are run
in full dose-response curves and the ECSO or ICSO values are calculated.
WO 00/66592 CA 02371642 2001-10-29
PCT/US00/11836
-36-
Table 3. Estimated ECSp,
standard error (SE),
and 95% confidence intervals
(CI) for individual studies
ECSO 95% CI
Compound Ex~ (nM~ SE lower upper
Trimegestone 1 0.017 0.003 0.007 0.040
2 0.014 0.001 0.011 0.017
3 0.019 0.001 0.016 0.024
MPA 1 0.019 0.001 0.013 0.027
2 0.017 0.001 0.011 0.024
Table 4. Estimated ICso, nd 95% confident
standard error, a interval
for the
antiprogestin, RU486
ICSO 95% CI
Compound Ex~ (nM) SE lower upper
RU486 1 0.011 0.001 0.008 0.014
2 0.016 0.001 0.014 0.020
3 0.018 0.001 0.014 0.022
ECS~: Concentration of a compound that gives half maximal increase in BrdU
incorporation with SE: ICSO: Concentration of a compound that gives half
maximal
decrease in 0.1 trimegestone induced BrdU incorporation with SE
4. T47D cell alkaline phos~hatase assay
The purpose of this assay is to identiy progestins or antiprogestins b~~
2~ determining a compound's erect on alkaline phosphatase activity in T47D
cells. The
materials and methods used in this assay are as follows.
a. Culture medium: DMEM:F12 (1:1) (GIBCO. BRL)
supplemented with 5°ro w/vj charcoal stripped fetal bovine serum (not
heat-inactivated),
100U/ml penicillin. 100 ~g/ml streptomycin. and 2 mM GlutaMax (GIBCO. BRL).
CA 02371642 2001-10-29
WO 00/66592 PCT/US00/11836
-37-
b. Alkaline phosphatase assay buffer:
I. 0.1 M Tris-HCI, pH 9.8, containing 0.2% Triton X-100
II. 0.1 M Tris-HCI, pH 9.8 containing 4 mM p-nitrophenyl phosphate
(Sigma).
c. Cell Culture and Treatment:
Frozen T47D cells were thawed in a 37°C water
bath and diluted to 280,000 cells/ml in culture medium. To each well in a 96-
well plate
(Falcon, Becton Dickinson Labware), 180 pl of diluted cell suspension was
added.
Twenty ~l of reference or test compounds diluted in the culture medium was
then added
to each well. When testing for progestin antagonist activity, reference
antiprogestins or
test compounds were added in the presence of 1 nM progesterone. The cells were
incubated at 37°C in a 5% COZ/humidified atmosphere for 24 hr.
d. Alkaline Phosphatase Enzyme Assay:
At the end of treatment, the medium was removed
from the plate and fifty p.l of assay buffer I was added to each well. The
plates were
shaken in a titer plate shaker for 15 min. Then 150 pl of assay buffer II was
added to
each well. Optical density measurements were taken at 5 min intervals for 30
min at a
test wavelength of 405 nM.
e. Analysis of Results: Analysis of dose-response data
For reference and test compounds. a dose response curve
is generated for dose (X-axis) vs. the rate of enzyme reaction (slope) (Y-
axis). Square
root-transformed data are used for analysis of variance and nonlinear dose
response
curve fitting for both agonist and antagonist modes. Huber weighting is used
to
downweight the effects of outliers. ECS~ or ICSO values are calculated from
the
retransformed values. JMP software (SAS Institute. Inc.) is used for both one-
way
analysis of variance and non-linear dose response analyses in both single dose
and dose
response studies.
WO 00/66592 CA 02371642 2001-l0-29 PCT/US00/11836
-38-
f. Reference Compounds:
Progesterone and trimegestone are reference progestins and RU486 is the
reference antiprogestin. All reference compounds are run in full dose response
curves
and the ECS~ or ICSO values are calculated.
Table 5. Estimated ECSp, standard error (SE), and 95% confidence intervals
(CI) for reference progestins from three independent experiments
EC50 95% CI
Compound Exp (nM) SE lower upper
Progesterone 1 0.839 0.030 0.706 0.996
2 0.639 0.006 0.611 0.669
3 1.286 0.029 1.158 1.429
Trimegestone 1 0.084 0.002 0.076 0.091
2 0.076 0.001 0.072 0.080
3 0.160 0.004 0.141 0.181
Table 6. Estimated ICSp, standard error, and 95% confident interval for the
reference antiprogestin RU486 from three independent experiments
IC 50 95% CI
Compound Ex~ (nM) SE lower upper
RU486 1 0.103 0.002 0.092 0.11
2 0.120 0.001 0.115 0.126
3 0.094 0.007 0.066 0.134
B. In-vivo Biology
The primary in-vivo assay is the rat decidualization model. which may be used
to determine progestational effects of both agonists and antagonists. The
secondan~ in-
vivo assay is the rat ovulation inhibition model, which is under development,
and hence
the protocol is un-available.
CA 02371642 2001-10-29
WO 00/66592 PCT/US00/11836
-39-
1. Rat decidualization assay The objective of this procedure is used to
evaluate the effect of progestins and antiprogestins on rat uterine
decidualization and
compare the relative potencies of various test compounds. The materials and
methods
used in this assay are as follows.
a. Methods: Test compounds are dissolved in 100% ethanol
and mixed with corn oil (vehicle). Stock solutions of the test compounds in
oil
(MazolaTM) are then prepared by heating (~80 oC) the mixture to evaporate
ethanol.
Test compounds are subsequently diluted with 100% corn oil or 10% ethanol in
corn
oil prior to the treatment of animals. No difference in decidual response was
found
when these two vehicles were compared.
b. Animals (RACUC~rotocol #5002)
Ovariectomized mature female Sprague-Dawley rats (~60-day old and 230g)
are obtained from Taconic (Taconic Farms, NY) following surgery. Ovariectomy
is
performed at least 10 days prior to treatment to reduce circulating sex
steroids.
Animals are housed under 12 hr light/dark cycle and given standard rat chow
and
water ad libitum.
c. Treatment
Rats are weighed and randomly assigned to groups of 4 or 5
before treatment. Test compounds in 0.2 ml vehicle are administered by
subcutaneous
injection in the nape of the neck or by gavage using 0.~ ml. The animals are
treated
once daily for seven days. For testing antiprogestins, animals are given the
test
compounds and a EC50 dose of progesterone (5.6 mg/kg) during the first three
days
of treatment. Following decidual stimulation, animals continue to receme
progesterone until necropsy four days later.
d. Dosin
Doses are prepared based upon mg/kg mean group body
weight. In all studies, a control group receiving vehicle is included.
Determination of
dose-response curves is carried out using doses with half log increases (e.g.
0.1. 0.3.
1Ø 3.0 mg/kg...).
WO 00/66592 CA 02371642 2001-10-29
PCT/US00/11836
-40-
e. Decidual induction
Approximately 24 hr after the third injection, decidualization
is induced in one of the uterine horns by scratching the antimesometrial
luminal
epithelium with a blunt 21 G needle. The contralateral horn is not scratched
and serves
as an unstimulated control. Approximately 24 hr following the final treatment,
rats are
sacrificed by C02 asphyxiation and body weight measured. Uteri are removed and
trimmed of fat. Decidualized (D-horn) and control (C-horn) uterine horns are
weighed
separately.
f Analysis of Results:
I O The increase in weight of the decidualized uterine horn is
calculated by D-horn/C-hom and logarithmic transformation is used to maximize
normality and homogeneity of variance. The Huber M-estimator is used to down
weight the outlying transformed observations for both dose-response curve
fitting and
one-way analysis of variance. JMP software (SAS Institute, Inc.) is used for
both one-
way ANOVA and non-linear dose-response analyses.
g. Reference Compounds:
All progestin reference compounds were run in full dose-
response curves and the ECSO for uterine wet weight were calculated.
Table 7. Estimated
ECso, standard error
(SE), and 95% confidence
intervals for
individual studies
ECSO 95% CI
Compound Exp (m~/k~ s SE lower upper
c )
Progesterone 1 5.50 0.77 4.21 7.20
2 6.21 1.12 4.41 8.76
3-Ketodesogestrel 1 0.11 0.02 0.07 0.16
0.10 0.05 0.11 0.25
3 0.06 0.03 0.03 0.14
WO 00/66592 CA 02371642 2001-10-29
PCT/t 1500/11836
-41 -
Levonorgestrel 1 0.08 0.03 0.04 0.16
2 0.12 0.02 0.09 0.17
3 0.09 0.02 0.06 0.13
4 0.09 0.02 0.06 0.14
MPA 1 0.42 0.03 0.29 0.60
2 0.39 0.05 0.22 0.67
3 0.39 0.04 0.25 0.61
Table 8. Estimated standardrror, and e
average ECSO~ e 95% confidenc
intervals for of 3
dose-response reference
curves compounds
EC50 95% CI
Compound (m~/k~ SE lower uuper
s c )
Progesterone 5.62 0.62 4.55 7.00
3-Ketodesogestrel0.10 0.02 0.07 0.14
Levonorgestrel 0.10 0.01 0.08 0.12
Table 9. Estimated ICso, standard error, and 95% confident interval for the
antiprogestin, RU 486
ICso 95% CI
Compound E~ (m~/k~ p o ) SE lower upper
RU 486 1 0.21 0.07 0.05 0.96
2 0.14 0.02 0.08 0.27
Concentration: Compound concentration in assay(default-mg/kg body weight)
Route of administration: Route the compound is administered to the animals
Bodv weight: Mean total animal bodv weight (default-kg)
D-horn: Wet weight of decidualized uterine horn (default-mg)
WO 00/66592 CA 02371642 2001-10-29 pCT/US00/11836
-42-
C-horn: Wet weight of control uterine horn (default-mg)
Decidual response: [(D-C)/C]x100%
Progestational activiy: Compounds that induce decidualization significantly
(p<0.05) compared to vehicle control are considered active
Antiprogestational activiri-: Compounds that decrease ECSO progesterone
induced decidualization significantly (p<0.05)
ECSO for uterine weight: Concentration of compound that gives half maximal
increase in decidual response (default-mg/kg)
ICSO for uterine weight: Concentration of compound that gives half maximal
decrease in ECSO progesterone induced decidual response (default-mg/kg)
All publications cited in this specification are incorporated herein by
reference herein. While the invention has been described with reference to a
particularly preferred embodiment, it will be appreciated that modifications
can be
made without departing from the spirit of the invention. Such modifications
are
intended to fall within the scope of the appended claims.
