U.S. patent application number 14/766632 was filed with the patent office on 2015-12-31 for modulators of methyl modifying enzymes, compositions and uses thereof.
This patent application is currently assigned to Constellation Pharmaceuticals, Inc.. The applicant listed for this patent is CONSTELLATION PHARMACEUTICALS, INC.. Invention is credited to Brian K. Albrecht, James Edmund Audia, Andrew S. Cook, Les A. Dakin, Martin Duplessis, Victor S. Gehling, Jean-Christophe Harmange, Christopher G. Nasveschuk, Rishi G. Vaswani.
Application Number | 20150376190 14/766632 |
Document ID | / |
Family ID | 48948171 |
Filed Date | 2015-12-31 |
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United States Patent
Application |
20150376190 |
Kind Code |
A1 |
Albrecht; Brian K. ; et
al. |
December 31, 2015 |
MODULATORS OF METHYL MODIFYING ENZYMES, COMPOSITIONS AND USES
THEREOF
Abstract
Agents having the structural Formula (II) for modulating histone
methyl modifying enzymes, compositions and uses thereof for
instance as anti-cancer agents are provided herein.
##STR00001##
Inventors: |
Albrecht; Brian K.;
(Cambridge, MA) ; Audia; James Edmund; (Cambridge,
MA) ; Cook; Andrew S.; (Stow, MA) ; Dakin; Les
A.; (Natick, MA) ; Duplessis; Martin;
(Somerville, MA) ; Gehling; Victor S.;
(Somerville, MA) ; Harmange; Jean-Christophe;
(Andover, MA) ; Nasveschuk; Christopher G.;
(Stoneham, MA) ; Vaswani; Rishi G.; (Lexington,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CONSTELLATION PHARMACEUTICALS, INC. |
Cambridge |
MA |
US |
|
|
Assignee: |
Constellation Pharmaceuticals,
Inc.
Cambridge
MA
|
Family ID: |
48948171 |
Appl. No.: |
14/766632 |
Filed: |
February 11, 2014 |
PCT Filed: |
February 11, 2014 |
PCT NO: |
PCT/US2014/015706 |
371 Date: |
August 7, 2015 |
Current U.S.
Class: |
514/265.1 ;
514/300; 544/280; 546/112 |
Current CPC
Class: |
C07D 471/04 20130101;
A61P 15/00 20180101; C07D 401/12 20130101; C07D 487/04 20130101;
A61P 11/00 20180101; A61P 17/00 20180101; A61P 13/12 20180101; A61P
25/00 20180101; C07D 405/14 20130101; C07D 417/14 20130101; C07D
417/04 20130101; A61P 1/04 20180101; A61P 1/16 20180101; A61P 43/00
20180101; C07D 409/06 20130101; A61P 13/10 20180101; C07D 401/14
20130101; A61P 13/08 20180101; A61P 35/00 20180101; A61K 31/4545
20130101 |
International
Class: |
C07D 487/04 20060101
C07D487/04; C07D 471/04 20060101 C07D471/04 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 11, 2013 |
US |
2013/025639 |
Claims
1. A compound having the structural Formula (II): ##STR00091## or a
pharmaceutically acceptable salt thereof, wherein: A is CH or N;
R.sup.1a is selected from --C.sub.1-C.sub.2 alkyl and
--O--(C.sub.1-C.sub.2 alkyl), wherein R.sup.1a is optionally
substituted with one or more fluoro; R.sup.4a is selected from
--(C.sub.1-C.sub.3 alkylene)-O--(C.sub.1-C.sub.2 alkyl),
1-substituted-pipieridin-4-yl, C.sub.3-C.sub.6 cycloalkyl
optionally substituted with one or more fluoro, and
tetrahydropyranyl; and R.sup.13 is selected from hydrogen, halo,
phenyl, pyridinyl, and --O--(C.sub.1-C.sub.4 alkyl).
2. The compound of claim 1, wherein R.sup.1a is selected from
--OCH.sub.3, --CH.sub.3, --OCHF.sub.2, and --CH.sub.2CH.sub.3.
3. The compound of claim 1 or 2, wherein the
1-substituted-pipieridin-4-yl is a
1-halo(C.sub.1-C.sub.3)alkyl-piperidin-4-yl.
4. The compound of any one of claims 1 to 3, wherein R.sup.4a is
selected from --CH.sub.2OCH.sub.3, --CH(CH.sub.3)OCH.sub.3,
4,4-difluorocyclohexyl, cyclopropyl, tetrayhyrdopyran-4-yl,
1-(t-butoxycarbonyl)-piperidin-4-yl,
1-(isobutoxycarbonyl)-piperidin-4-yl,
1-(isopropoxycarbonyl)-piperidin-4-yl,
1-(2-fluoroethyl)-piperidin-4-yl,
1-(2,2-difluoroethyl)-piperidin-4-yl,
1-(2,2,2-trifluoroethyl)-piperidin-4-yl,
1-(2-hydroxyisobutyl)-piperidin-4-yl,
1-(hydroxyisopropylcarbonyl)-piperidin-4-yl,
1-(ethoxycarbonylmethyl)-piperidin-4-yl,
1-(isopropylcarbonyl)-piperidin-4-yl, 1-methylpiperidin-4-yl,
1-(methylsulfonyl)-piperidin-4-yl,
1-(ethylsulfonyl)-piperidin-4-yl,
1-(isopropylsulfonyl)-piperidin-4-yl, 1-(phenyl)-piperidin-4-yl,
1-(oxetan-3-yl)piperidin-4-yl, 1-(pyridin-2-yl)-piperidin-4-yl, and
1-(pyrimidin-2-yl)-piperidin-4-yl.
5. The compound of any one of claims 1 to 4, wherein R.sup.13 is
selected from hydrogen, chloro, fluoro, --OCH(CH.sub.3).sub.2,
phenyl, and pyridin-2-yl.
6. The compound of claim 1, wherein the compound is
N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methyl-1-(-
1-(1-(2,2,2-trifluoroethyl)piperidin-4-yl)ethyl)-1H-indole-3-carboxamide,
or a pharmaceutically acceptable salt thereof.
7. The compound of claim 6, wherein the compound is
R--N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methyl--
1-(1-(1-(2,2,2-trifluoroethyl)piperidin-4-yl)ethyl)-1H-indole-3-carboxamid-
e, or a pharmaceutically acceptable salt thereof.
8. The compound of claim 1, wherein the compound is
1-(1-(1-(2,2-difluoroethyl)piperidin-4-yl)ethyl)-N-((4-methoxy-6-methyl-2-
-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methyl-1H-indole-3-carboxamide,
or a pharmaceutically acceptable salt thereof.
9. The compound of claim 8, wherein the compound is
R-1-(1-(1-(2,2-difluoroethyl)piperidin-4-yl)ethyl)-N-((4-methoxy-6-methyl-
-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methyl-1H-indole-3-carboxamide,
or a pharmaceutically acceptable salt thereof.
10. The compound of claim 1, wherein the compound is
N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-1-(1-(1-(2-hydroxy-
-2-methylpropyl)piperidin-4-yl)ethyl)-2-methyl-1H-indole-3-carboxamide,
or a pharmaceutically acceptable salt thereof.
11. The compound of claim 10, wherein the compound is
R--N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-1-(1-(1-(2-hydr-
oxy-2-methylpropyl)piperidin-4-yl)ethyl)-2-methyl-1H-indole-3-carboxamide,
or a pharmaceutically acceptable salt thereof.
12. The compound of claim 1, wherein the compound is
1-(1-(1-ethylpiperidin-4-yl)ethyl)-N-((4-methoxy-6-methyl-2-oxo-1,2-dihyd-
ropyridin-3-yl)methyl)-2-methyl-1H-indole-3-carboxamide, or a
pharmaceutically acceptable salt thereof.
13. The compound of claim 12, wherein the compound is
R-1-(1-(1-ethylpiperidin-4-yl)ethyl)-N-((4-methoxy-6-methyl-2-oxo-1,2-dih-
ydropyridin-3-yl)methyl)-2-methyl-1H-indole-3-carboxamide, or a
pharmaceutically acceptable salt thereof.
14. A pharmaceutical composition comprising a compound of any one
of claims 1 to 13, or a pharmaceutically acceptable salt thereof;
and a pharmaceutically acceptable carrier.
15. Use of a compound of any one of claims 1 to 13, or a
pharmaceutically acceptable salt thereof, for the preparation of a
medicament for treating a disease or disorder associated with
cellular proliferation.
16. The use of claim 15, wherein the disease is cancer.
17. The use of claim 16, wherein the cancer is selected from breast
cancer, prostate cancer, colon cancer, renal cell carcinoma,
glioblastoma multiforme cancer, bladder cancer, melanoma, bronchial
cancer, lymphoma, and liver cancer.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to International
Application No. PCT/US2013/025639, filed Feb. 11, 2013.
International Application No. PCT/US2013/025639 claims priority to
U.S. Provisional Application Ser. Nos. 61/597,695, filed Feb. 10,
2012, and 61/667,821, filed Jul. 3, 2012. The entire contents of
the aforementioned applications are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] Eukaryotic chromatin is composed of macromolecular complexes
called nucleosomes. A nucleosome has 147 base pairs of DNA wrapped
around a protein octamer having two subunits of each of histone
protein H2A, H2B, H3, and H4. Histone proteins are subject to
post-translational modifications which in turn affect chromatin
structure and gene expression. One type of post-translational
modification found on histones is methylation of lysine and
arginine residues. Histone methylation plays a critical role in the
regulation of gene expression in eukaryotes. Methylation affects
chromatin structure and has been linked to both activation and
repression of transcription (Zhang and Reinberg, Genes Dev.
15:2343-2360, 2001). Enzymes that catalyze attachment and removal
of methyl groups from histones are implicated in gene silencing,
embryonic development, cell proliferation, and other processes.
SUMMARY OF THE INVENTION
[0003] The present disclosure encompasses the recognition that
methyl modifying enzymes are an attractive target for modulation,
given their role in the regulation of diverse biological processes.
It has now been found that compounds of this invention, and
pharmaceutically acceptable compositions thereof, are effective as
agents that stimulate activity of histone methyl modifying enzymes,
including histone methylases and histone demethylases. Such
compounds have the general formula II:
##STR00002##
or a pharmaceutically acceptable salt thereof, wherein each
variable is as defined herein.
[0004] Compounds of the present invention, and pharmaceutically
acceptable compositions thereof, are useful for treating a variety
of diseases, disorders or conditions, associated with a methyl
modifying enzyme. Such diseases, disorders, or conditions include
those described herein.
[0005] Compounds provided by this invention are also useful for the
study of methyl modifying enzymes in biological and pathological
phenomena; the study of intracellular signal transduction pathways
mediated by methyl modifying enzymes and the comparative evaluation
of new methyl modifying enzyme modulators.
BRIEF DESCRIPTION OF THE DRAWINGS
[0006] FIG. 1. Exemplary compounds of Formula II.
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
1. General Description of Compounds of the Invention
[0007] In certain embodiments, the present invention provides a
compound of Formula II:
##STR00003##
or a pharmaceutically acceptable salt thereof, wherein:
[0008] A is CH or N;
[0009] R.sup.1a is selected from --C.sub.1-C.sub.2 alkyl and
--O--(C.sub.1-C.sub.2 alkyl), wherein R.sup.1a is optionally
substituted with one or more fluoro;
[0010] R.sup.4a is selected from --(C.sub.1-C.sub.4
alkylene)-O--(C.sub.1-C.sub.3 alkyl),
1-substituted-pipieridin-4-yl, C.sub.3-C.sub.6 cycloalkyl
optionally substituted with one or more fluoro, and
tetrahydropyranyl; and
[0011] R.sup.13 is selected from hydrogen, halo, phenyl, pyridinyl,
and --O--(C.sub.1-C.sub.4 alkyl).
2. Compounds and Definitions
[0012] Definitions of specific functional groups and chemical terms
are described in more detail below. For purposes of this invention,
the chemical elements are identified in accordance with the
Periodic Table of the Elements, CAS version, Handbook of Chemistry
and Physics, 75.sup.th Ed., inside cover, and specific functional
groups are generally defined as described therein. Additionally,
general principles of organic chemistry, as well as specific
functional moieties and reactivity, are described in Organic
Chemistry, Thomas Sorrell, University Science Books, Sausalito,
1999; Smith and March March's Advanced Organic Chemistry, 5.sup.th
Edition, John Wiley & Sons, Inc., New York, 2001; Larock,
Comprehensive Organic Transformations, VCH Publishers, Inc., New
York, 1989; Carruthers, Some Modern Methods of Organic Synthesis,
3.sup.rd Edition, Cambridge University Press, Cambridge, 1987; the
entire contents of each of which are incorporated herein by
reference.
[0013] Unless otherwise stated, structures depicted herein are also
meant to include all isomeric (e.g., enantiomeric, diastereomeric,
and geometric (or conformational)) forms of the structure; for
example, the R and S configurations for each asymmetric center, Z
and E double bond isomers, and Z and E conformational isomers.
Therefore, single stereochemical isomers as well as enantiomeric,
diastereomeric, and geometric (or conformational) mixtures of the
present compounds are within the scope of the invention. Unless
otherwise stated, all tautomeric forms of the compounds of the
invention are within the scope of the invention. Additionally,
unless otherwise stated, structures depicted herein are also meant
to include compounds that differ only in the presence of one or
more isotopically enriched atoms. For example, compounds having the
present structures including the replacement of hydrogen by
deuterium or tritium, or the replacement of a carbon by a .sup.13C-
or .sup.14C-enriched carbon are within the scope of this invention.
Such compounds are useful, for example, as analytical tools, as
probes in biological assays, or as therapeutic agents in accordance
with the present invention.
[0014] Where a particular enantiomer is preferred, it may, in some
embodiments be provided substantially free of the corresponding
enantiomer, and may also be referred to as "optically enriched."
"Optically-enriched," as used herein, means that the compound is
made up of a significantly greater proportion of one enantiomer. In
certain embodiments the compound is made up of at least about 90%
by weight of a preferred enantiomer. In other embodiments the
compound is made up of at least about 95%, 98%, or 99% by weight of
a preferred enantiomer. Preferred enantiomers may be isolated from
racemic mixtures by any method known to those skilled in the art,
including chiral high pressure liquid chromatography (HPLC) and the
formation and crystallization of chiral salts or prepared by
asymmetric syntheses. See, for example, Jacques et al.,
Enantiomers, Racemates and Resolutions (Wiley Interscience, New
York, 1981); Wilen, et al., Tetrahedron 33:2725 (1977); Eliel, E.
L. Stereochemistry of Carbon Compounds (McGraw-Hill, N Y, 1962);
Wilen, S. H. Tables of Resolving Agents and Optical Resolutions p.
268 (E. L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, Ind.
1972).
[0015] A wavy bond () at a chiral center in a chemical structure is
used to denote compounds of the invention that are optically pure,
but whose optical rotation has not been determined. A straight bond
at a chiral center indicates a racemic mixture although, as stated
above, the invention also includes all possible isomeric forms of
the racemate.
[0016] The terms "halo" and "halogen" as used herein refer to an
atom selected from fluorine (fluoro,--F), chlorine (chloro,--Cl),
bromine (bromo,--Br), and iodine (iodo,--I).
[0017] The term "alkylene" refers to a bivalent alkyl group. An
"alkylene chain" is a polymethylene group, i.e.,
--(CH.sub.2).sub.n--, wherein n is a positive integer, preferably
from 1 to 6, from 1 to 4, from 1 to 3, from 1 to 2, or from 2 to 3.
A substituted alkylene chain is a polymethylene group in which one
or more methylene hydrogen atoms are replaced with a substituent.
Suitable substituents include those described below for a
substituted aliphatic group.
[0018] The term "alkyl," as used herein, refers to a monovalent
saturated, straight- or branched-chain hydrocarbon radical derived
from an aliphatic moiety containing between one and six carbon
atoms by removal of a single hydrogen atom. In some embodiments,
alkyl contains 1-5 carbon atoms. In another embodiment, alkyl
contains 1-4 carbon atoms. In still other embodiments, alkyl
contains 1-3 carbon atoms. In yet another embodiment, alkyl
contains 1-2 carbons. Examples of alkyl radicals include, but are
not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl,
iso-butyl, sec-butyl, sec-pentyl, iso-pentyl, tert-butyl, n-pentyl,
neopentyl, n-hexyl, sec-hexyl, and the like.
[0019] As described herein, compounds of the invention may contain
"optionally substituted" moieties. In general, the term
"substituted", whether preceded by the term "optionally" or not,
means that one or more hydrogens of the designated moiety are
replaced with a suitable substituent. Unless otherwise indicated,
an "optionally substituted" group may have a suitable substituent
at each substitutable position of the group, and when more than one
position in any given structure may be substituted with more than
one substituent selected from a specified group, the substituent
may be either the same or different at each position. Combinations
of substituents envisioned under this invention are preferably
those that result in the formation of stable or chemically feasible
compounds. The term "stable", as used herein, refers to compounds
that are not substantially altered when subjected to conditions to
allow for their production, detection, and, in certain embodiments,
their recovery, purification, and use for one or more of the
purposes disclosed herein.
[0020] Suitable monovalent substituents on a substitutable carbon
atom of an "optionally substituted" group are independently
halogen; --(CH.sub.2).sub.0-4R.sup..smallcircle.;
--(CH.sub.2).sub.0-4OR.sup..smallcircle.;
--O--(CH.sub.2).sub.0-4C(O)OR.sup..smallcircle.;
--(CH.sub.2).sub.0-4CH(OR.sup..smallcircle.).sub.2;
--(CH.sub.2).sub.0-4SR.sup..smallcircle.; --(CH.sub.2).sub.0-4Ph,
which may be substituted with R.sup..smallcircle.;
--(CH.sub.2).sub.0-4O(CH.sub.2).sub.0-1Ph which may be substituted
with R.sup..smallcircle.; --CH.dbd.CHPh, which may be substituted
with R.sup..smallcircle.; --NO.sub.2; --CN; --N.sub.3;
--(CH.sub.2).sub.0-4N(R.sup..smallcircle.).sub.2;
--(CH.sub.2).sub.0-4N(R.sup..smallcircle.)C(O)R.sup..smallcircle.;
--N(R.sup..smallcircle.)C(S)R.sup..smallcircle.;
--(CH.sub.2).sub.0-4N(R.sup..smallcircle.)C(O)NR.sup..smallcircle..sub.2;
--N(R.sup..smallcircle.)C(S)NR.sup..smallcircle..sub.2;
--(CH.sub.2).sub.0-4N(R.sup..smallcircle.)C(O)OR.sup..smallcircle.;
--N(R.sup..smallcircle.)N(R.sup..smallcircle.)C(O)R.sup..smallcircle.;
--N(R.sup..smallcircle.)N(R.sup..smallcircle.)C(O)NR.sup..smallcircle..su-
b.2; --N(R.sup..smallcircle.)
N(R.sup..smallcircle.)C(O)OR.sup..smallcircle.;
--(CH.sub.2).sub.0-4C(O)R; --C(S)R.sup..smallcircle.;
--(CH.sub.2).sub.0-4C(O)OR.sup..smallcircle.;
--(CH.sub.2).sub.0-4C(O)SR.sup..smallcircle.;
--(CH.sub.2).sub.0-4C(O)OSiR.sup..smallcircle..sub.3;
--(CH.sub.2).sub.0-4OC(O)R.sup..smallcircle.;
--OC(O)(CH.sub.2).sub.0-4SR--; --SC(S)SR.sup..smallcircle.;
--(CH.sub.2).sub.0-4SC(O)R.sup..smallcircle.;
--(CH.sub.2).sub.0-4C(O)NR.sup..smallcircle..sub.2;
--C(S)NR.sup..smallcircle..sub.2; --C(S)SR.sup..smallcircle.;
--SC(S)SR.sup..smallcircle.;
--(CH.sub.2).sub.0-4OC(O)NR.sup..smallcircle..sub.2;
--C(O)N(OR.sup..smallcircle.) R.sup..smallcircle.;
--C(O)C(O)R.sup..smallcircle.;
--C(O)CH.sub.2C(O)R.sup..smallcircle.;
--C(NOR.sup..smallcircle.)R.sup..smallcircle.;
--(CH.sub.2).sub.0-4SSR.sup..smallcircle.;
--(CH.sub.2).sub.0-4S(O).sub.2R.sup..smallcircle.;
--(CH.sub.2).sub.0-4S(O).sub.2OR.sup..smallcircle.;
--(CH.sub.2).sub.0-4OS(O).sub.2R.sup..smallcircle.;
--S(O).sub.2NR.sup..smallcircle..sub.2;
--(CH.sub.2).sub.0-4S(O)R.sup..smallcircle.;
--N(R.sup..smallcircle.)S(O).sub.2NR.sup..smallcircle..sub.2;
--N(R.sup..smallcircle.)S(O).sub.2R.sup..smallcircle.;
--N(OR.sup..smallcircle.)R.sup..smallcircle.;
--C(NH)NR.sup..smallcircle..sub.2; --P(O).sub.2R.sup..smallcircle.;
--P(O)R.sup..smallcircle..sub.2; --OP(O)R.sup..smallcircle..sub.2;
--OP(O)(OR.sup..smallcircle.).sub.2; --SiR.sup..smallcircle..sub.3;
--(C.sub.1-4 straight or branched
alkylene)O--N(R.sup..smallcircle.).sub.2; or --(C.sub.1-4 straight
or branched alkylene)C(O)O--N(R.sup..smallcircle.).sub.2, wherein
each R.sup..smallcircle. may be substituted as defined below and is
independently hydrogen, C.sub.1-6 aliphatic, --CH.sub.2Ph,
--O(CH.sub.2).sub.0-1Ph, or a 5-6-membered saturated, partially
unsaturated, or aryl ring having 0-4 heteroatoms independently
selected from nitrogen, oxygen, or sulfur, or, notwithstanding the
definition above, two independent occurrences of
R.sup..smallcircle., taken together with their intervening atom(s),
form a 3-12-membered saturated, partially unsaturated, or aryl
mono- or bicyclic ring having 0-4 heteroatoms independently
selected from nitrogen, oxygen, or sulfur, which may be substituted
as defined below.
[0021] Suitable substituents on a substitutable nitrogen of an
"optionally substituted" group include --R.sup..dagger.,
--NR.sup..dagger..sub.2, --C(O)R.sup..dagger.,
--C(O)OR.sup..dagger., --C(O)C(O)R.sup..dagger.,
--C(O)CH.sub.2C(O)R.sup..dagger., --S(O).sub.2R.sup..dagger.,
--S(O).sub.2NR.sup..dagger..sub.2, --C(S)NR.sup..dagger..sub.2,
--C(NH)NR.sup..dagger..sub.2, or
--N(R.sup..dagger.)S(O).sub.2R.sup..dagger.; wherein each
R.sup..dagger. is independently hydrogen, C.sub.1-6 aliphatic which
may be substituted as defined below, unsubstituted --OPh, or an
unsubstituted 5-6-membered saturated, partially unsaturated, or
aryl ring having 0-4 heteroatoms independently selected from
nitrogen, oxygen, or sulfur, or, notwithstanding the definition
above, two independent occurrences of R.sup..dagger., taken
together with their intervening atom(s) form an unsubstituted
3-12-membered saturated, partially unsaturated, or aryl mono- or
bicyclic ring having 0-4 heteroatoms independently selected from
nitrogen, oxygen, or sulfur.
[0022] Suitable substituents on the aliphatic group of
R.sup..dagger. are independently halogen, --R.sup..cndot.,
-(haloR.sup..cndot.), --OH, --OR.sup..cndot.,
--O(haloR.sup..cndot.), --CN, --C(O)OH, --C(O)OR.sup..cndot.,
--NH.sub.2, --NHR.sup..cndot., --NR.sup..cndot..sub.2, or
--NO.sub.2, wherein each R.sup..cndot. is unsubstituted or where
preceded by "halo" is substituted only with one or more halogens,
and is independently C.sub.1-4aliphatic, --CH.sub.2Ph,
--O(CH.sub.2).sub.0-1Ph, or a 5-6-membered saturated, partially
unsaturated, or aryl ring having 0-4 heteroatoms independently
selected from nitrogen, oxygen, or sulfur.
[0023] As used herein, the term "inhibitor" is defined as a
compound that binds to and/or inhibits a target
S-adenosylmethionine (SAM) utilizing enzyme with measurable
affinity. In certain embodiments, an inhibitor has an IC.sub.50
and/or binding constant of less about 50 .mu.M, less than about 1
.mu.M, less than about 500 nM, less than about 100 nM, or less than
about 10 nM.
[0024] The terms "measurable affinity" and "measurably inhibit," as
used herein, means a measurable change in activity of at least one
SAM utilizing enzyme between a sample comprising a provided
compound, or composition thereof, and at least one SAM dependent
enzyme, and an equivalent sample comprising at least one SAM
dependent enzyme, in the absence of said compound, or composition
thereof.
3. Description of Exemplary Compounds
[0025] In some embodiments of Formula II, R.sup.1a is selected from
--OCH.sub.3, --CH.sub.3, --OCHF.sub.2, and --CH.sub.2CH.sub.3.
[0026] In some embodiments of Formula II, R.sup.4a is selected from
--CH.sub.2OCH.sub.3, --CH(CH.sub.3)OCH.sub.3,
4,4-difluorocyclohexyl, cyclopropyl, tetrayhyrdopyran-4-yl,
1-(t-butoxycarbonyl)-piperidin-4-yl,
1-(isobutoxycarbonyl)-piperidin-4-yl,
1-(isopropoxycarbonyl)-piperidin-4-yl,
1-(2-fluoroethyl)-piperidin-4-yl,
1-(2,2-difluoroethyl)-piperidin-4-yl,
1-(2,2,2-trifluoroethyl)-piperidin-4-yl,
1-(2-hydroxyisobutyl)-piperidin-4-yl,
1-(hydroxyisopropylcarbonyl)-piperidin-4-yl,
1-(ethoxycarbonylmethyl)-piperidin-4-yl,
1-(isopropylcarbonyl)-piperidin-4-yl, 1-methylpiperidin-4-yl,
1-(methylsulfonyl)-piperidin-4-yl,
1-(ethylsulfonyl)-piperidin-4-yl,
1-(isopropylsulfonyl)-piperidin-4-yl, 1-(phenyl)-piperidin-4-yl,
1-(oxetan-3-yl)piperidin-4-yl, 1-(pyridin-2-yl)-piperidin-4-yl, and
1-(pyrimidin-2-yl)-piperidin-4-yl.
[0027] In some embodiments of Formula II, R.sup.13 is selected from
hydrogen, chloro, fluoro, --OCH(CH.sub.3).sub.2, phenyl, and
pyridin-2-yl.
[0028] Exemplary compounds of Formula II are set forth in FIG. 1.
In some cases two (or more) of the compounds in FIG. 1 having one
(or more) wavy bonds will have the exact same structure. Because
the wavy bond represents a chiral center of undetermined optical
rotation, such compounds will be understood to be separate and
distinct optical isomers of one another. FIG. 1 is annotated to
indicate those sets of two or more compounds that have the same
depicted structure, but are of different stereochemistry.
4. Uses, Formulation and Administration
Pharmaceutically Acceptable Compositions
[0029] According to another embodiment, the invention provides a
composition comprising a compound of this invention or a
pharmaceutically acceptable derivative thereof and a
pharmaceutically acceptable carrier, adjuvant, or vehicle. The
amount of compound in compositions of this invention is such that
is effective to measurably modulate a histone methyl modifying
enzyme, or a mutant thereof, in a biological sample or in a
patient. In certain embodiments, the amount of compound in
compositions of this invention is such that is effective to
measurably modulate a histone methyl modifying enzyme, or a mutant
thereof, in a biological sample or in a patient.
[0030] In certain embodiments, a composition of this invention is
formulated for administration to a patient in need of such
composition. In some embodiments, a composition of this invention
is formulated for oral administration to a patient.
[0031] The term "patient," as used herein, means an animal,
preferably a mammal, and most preferably a human.
[0032] The term "pharmaceutically acceptable carrier, adjuvant, or
vehicle" refers to a non-toxic carrier, adjuvant, or vehicle that
does not destroy the pharmacological activity of the compound with
which it is formulated. Pharmaceutically acceptable carriers,
adjuvants or vehicles that may be used in the compositions of this
invention include, but are not limited to, ion exchangers, alumina,
aluminum stearate, lecithin, serum proteins, such as human serum
albumin, buffer substances such as phosphates, glycine, sorbic
acid, potassium sorbate, partial glyceride mixtures of saturated
vegetable fatty acids, water, salts or electrolytes, such as
protamine sulfate, disodium hydrogen phosphate, potassium hydrogen
phosphate, sodium chloride, zinc salts, colloidal silica, magnesium
trisilicate, polyvinyl pyrrolidone, cellulose-based substances,
polyethylene glycol, sodium carboxymethylcellulose, polyacrylates,
waxes, polyethylene-polyoxypropylene-block polymers, polyethylene
glycol and wool fat.
[0033] A "pharmaceutically acceptable derivative" means any
non-toxic salt, ester, salt of an ester or other derivative of a
compound of this invention that, upon administration to a
recipient, is capable of providing, either directly or indirectly,
a compound of this invention or an inhibitorily active metabolite
or residue thereof.
[0034] Compositions of the present invention may be administered
orally, parenterally, by inhalation spray, topically, rectally,
nasally, buccally, vaginally or via an implanted reservoir. The
term "parenteral" as used herein includes subcutaneous,
intravenous, intramuscular, intra-articular, intra-synovial,
intrasternal, intrathecal, intrahepatic, intralesional and
intracranial injection or infusion techniques. Preferably, the
compositions are administered orally, intraperitoneally or
intravenously. Sterile injectable forms of the compositions of this
invention may be aqueous or oleaginous suspension. These
suspensions may be formulated according to techniques known in the
art using suitable dispersing or wetting agents and suspending
agents. The sterile injectable preparation may also be a sterile
injectable solution or suspension in a non-toxic parenterally
acceptable diluent or solvent, for example as a solution in
1,3-butanediol. Among the acceptable vehicles and solvents that may
be employed are water, Ringer's solution and isotonic sodium
chloride solution. In addition, sterile, fixed oils are
conventionally employed as a solvent or suspending medium.
[0035] For this purpose, any bland fixed oil may be employed
including synthetic mono- or di-glycerides. Fatty acids, such as
oleic acid and its glyceride derivatives are useful in the
preparation of injectables, as are natural
pharmaceutically-acceptable oils, such as olive oil or castor oil,
especially in their polyoxyethylated versions. These oil solutions
or suspensions may also contain a long-chain alcohol diluent or
dispersant, such as carboxymethyl cellulose or similar dispersing
agents that are commonly used in the formulation of
pharmaceutically acceptable dosage forms including emulsions and
suspensions. Other commonly used surfactants, such as Tweens, Spans
and other emulsifying agents or bioavailability enhancers which are
commonly used in the manufacture of pharmaceutically acceptable
solid, liquid, or other dosage forms may also be used for the
purposes of formulation.
[0036] Pharmaceutically acceptable compositions of this invention
may be orally administered in any orally acceptable dosage form
including, but not limited to, capsules, tablets, aqueous
suspensions or solutions. In the case of tablets for oral use,
carriers commonly used include lactose and corn starch. Lubricating
agents, such as magnesium stearate, are also typically added. For
oral administration in a capsule form, useful diluents include
lactose and dried cornstarch. When aqueous suspensions are required
for oral use, the active ingredient is combined with emulsifying
and suspending agents. If desired, certain sweetening, flavoring or
coloring agents may also be added.
[0037] Alternatively, pharmaceutically acceptable compositions of
this invention may be administered in the form of suppositories for
rectal administration. These can be prepared by mixing the agent
with a suitable non-irritating excipient that is solid at room
temperature but liquid at rectal temperature and therefore will
melt in the rectum to release the drug. Such materials include
cocoa butter, beeswax and polyethylene glycols.
[0038] Pharmaceutically acceptable compositions of this invention
may also be administered topically, especially when the target of
treatment includes areas or organs readily accessible by topical
application, including diseases of the eye, the skin, or the lower
intestinal tract. Suitable topical formulations are readily
prepared for each of these areas or organs.
[0039] Topical application for the lower intestinal tract can be
effected in a rectal suppository formulation (see above) or in a
suitable enema formulation. Topically-transdermal patches may also
be used.
[0040] For topical applications, provided pharmaceutically
acceptable compositions may be formulated in a suitable ointment
containing the active component suspended or dissolved in one or
more carriers. Carriers for topical administration of compounds of
this invention include, but are not limited to, mineral oil, liquid
petrolatum, white petrolatum, propylene glycol, polyoxyethylene,
polyoxypropylene compound, emulsifying wax and water.
Alternatively, provided pharmaceutically acceptable compositions
can be formulated in a suitable lotion or cream containing the
active components suspended or dissolved in one or more
pharmaceutically acceptable carriers. Suitable carriers include,
but are not limited to, mineral oil, sorbitan monostearate,
polysorbate 60, cetyl esters wax, cetearyl alcohol,
2-octyldodecanol, benzyl alcohol and water.
[0041] For ophthalmic use, provided pharmaceutically acceptable
compositions may be formulated as micronized suspensions in
isotonic, pH adjusted sterile saline, or, preferably, as solutions
in isotonic, pH adjusted sterile saline, either with or without a
preservative such as benzylalkonium chloride. Alternatively, for
ophthalmic uses, the pharmaceutically acceptable compositions may
be formulated in an ointment such as petrolatum.
[0042] Pharmaceutically acceptable compositions of this invention
may also be administered by nasal aerosol or inhalation. Such
compositions are prepared according to techniques well-known in the
art of pharmaceutical formulation and may be prepared as solutions
in saline, employing benzyl alcohol or other suitable
preservatives, absorption promoters to enhance bioavailability,
fluorocarbons, and/or other conventional solubilizing or dispersing
agents.
[0043] Most preferably, pharmaceutically acceptable compositions of
this invention are formulated for oral administration. Such
formulations may be administered with or without food. In some
embodiments, pharmaceutically acceptable compositions of this
invention are administered without food. In other embodiments,
pharmaceutically acceptable compositions of this invention are
administered with food.
[0044] The amount of compounds of the present invention that may be
combined with the carrier materials to produce a composition in a
single dosage form will vary depending upon the host treated and
the particular mode of administration. Preferably, provided
compositions should be formulated so that a dosage of between
0.01-100 mg/kg body weight/day of the inhibitor can be administered
to a patient receiving these compositions.
[0045] It should also be understood that a specific dosage and
treatment regimen for any particular patient will depend upon a
variety of factors, including the activity of the specific compound
employed, the age, body weight, general health, sex, diet, time of
administration, rate of excretion, drug combination, and the
judgment of the treating physician and the severity of the
particular disease being treated. The amount of a compound of the
present invention in the composition will also depend upon the
particular compound in the composition.
Uses of Compounds and Pharmaceutically Acceptable Compositions
[0046] Compounds and compositions described herein are generally
useful for the modulating of activity of one or more enzymes
involved in epigenetic regulation.
[0047] Epigenetics is the study of heritable changes in gene
expression caused by mechanisms other than changes in the
underlying DNA sequence. Molecular mechanisms that play a role in
epigenetic regulation include DNA methylation and chromatin/histone
modifications. Histone methylation, in particular, is critical in
many epigenetic phenomena.
[0048] Chromatin, the organized assemblage of nuclear DNA and
histone proteins, is the basis for a multitude of vital nuclear
processes including regulation of transcription, replication,
DNA-damage repair and progression through the cell cycle. A number
of factors, such as chromatin-modifying enzymes, have been
identified that play an important role in maintaining the dynamic
equilibrium of chromatin (Margueron, et al. (2005) Curr. Opin.
Genet. Dev. 15:163-176).
[0049] Histones are the chief protein components of chromatin. They
act as spools around which DNA winds, and they play a role in gene
regulation. There are a total of six classes of histones (H1, H2A,
H2B, H3, H4, and H5) organized into two super classes: core
histones (H2A, H2B, H3, and H4) and linker histones (H1 and H5).
The basic unit of chromatin is the nucleosome, which consists of
about 147 base pairs of DNA wrapped around the histone octamer,
consisting of two copies each of the core histones H2A, H2B, H3,
and H4 (Luger, et al. (1997) Nature 389:251-260).
[0050] Histones, particularly residues of the amino termini of
histones H3 and H4 and the amino and carboxyl termini of histones
H2A, H2B and H1, are susceptible to a variety of post-translational
modifications including acetylation, methylation, phosphorylation,
ribosylation, sumoylation, ubiquitination, citrullination,
deimination, and biotinylation. The core of histones H2A and H3 can
also be modified. Histone modifications are integral to diverse
biological processes such as gene regulation, DNA repair, and
chromosome condensation.
[0051] The present disclosure provides compounds and compositions
for modulating activity of histone methyl modifying enzymes.
Histone methyl modifying enzymes are key regulators of cellular and
developmental processes. Histone methyl modifying enzymes may be
characterized as either histone methyl transferases or histone
demethylases. Histone demethylase enzymes have modules that mediate
binding to methylated residues. For example, multiple demethylases
contain a Tudor domain (e.g., JMJD2C/GASC1) or a PHD domain (e.g.,
JARID1C/SMCX, PHF8).
[0052] The lysine specificities of many histone methyltransferases
have been characterized. For example SET7/9, SMYD3, and MLL1-5 are
specific for H3K4. SUV39H1, DIM-5, and G9a are specific for H3K9.
SET8 is specific for H4K20.
[0053] DOT1 is an example of a non-SET domain containing histone
methylase. DOT1 methylates H3 on lysine 79.
[0054] Just as histone methylases have been shown to regulate
transcriptional activity, chromatin structure, and gene silencing,
demethylases have also been discovered which impact gene
expression. LSD1 was the first histone lysine demethylase to be
characterized. This enzyme displays homology to FAD-dependent amine
oxidases and acts as a transcriptional corepressor of neuronal
genes (Shi et al., Cell 119:941-953, 2004). Additional demethylases
defining separate demethylase families have been discovered,
including JHDM1 (or KDM2), JHDM2 (or KDM3), JMJD2 (or KDM4), JARID
(or KDM5), JMJD3 (or KDM6), and JMJD6 families (Lan et al., Curr.
Opin. Cell Biol. 20(3):316-325, 2008).
[0055] Demethylases act on specific lysine residues within
substrate sequences and discriminate between the degree of
methylation present on a given residue. For example, LSD1 removes
mono- or dimethyl-groups from H3K4. Members of the JARID1A-D family
remove trimethyl groups from H3K4. UTX and JMJD3 demethylate H3K27,
counteracting effects of EZH2 methylase activity. Substrate
specificities of other demethylases have been characterized (see
Shi, Nat. Rev. 8:829-833, 2007).
[0056] One class of histone methylases is characterized by the
presence of a SET domain, named after proteins that share the
domain, Su(var)3-9, enhancer of zeste [E(Z)], and trithorax. A SET
domain includes about 130 amino acids. SET domain-containing
methylase families include SUV39H1, SET1, SET2, EZH2, RIZ1, SMYD3,
SUV4-20H1, SET7/9, and PR-SET7/SET8 families (reviewed in Dillon et
al., Genome Biol. 6:227, 2005). Members of a family typically
include similar sequence motifs in the vicinity of and within the
SET domain. The human genome encodes over 50 SET domain-containing
histone protein methylases, any of which can be used in an assay
described herein.
[0057] EZH2 is an example of a human SET-domain containing
methylase. EZH2 associates with EED (Embryonic Ectoderm
Development) and SUZ12 (suppressor of zeste 12 homolog) to form a
complex known as PRC2 (Polycomb Group Repressive Complex 2) having
the ability to tri-methylate histone H3 at lysine 27 (Cao and
Zhang, Mol. Cell 15:57-67, 2004). PRC2 complexes can also include
RBAP46 and RBAP48 subunits.
[0058] The oncogenic activities of EZH2 have been shown by a number
of studies. In cell line experiments, over-expression of EZH2
induces cell invasion, growth in soft agar, and motility while
knockdown of EZH2 inhibits cell proliferation and cell invasion
(Kleer et al., 2003, Proc. Nat. Acad. Sci. USA 100:11606-11611;
Varambally et al., (2002), "The polycomb group protein EZH2 is
involved in progression of prostate cancer," Nature 419, 624-629).
It has been shown that EZH2 represses the expression of several
tumor suppressors, including E-cadherin, DAB2IP and RUNX3 among
others. In xenograft models, EZH2 knockdown inhibits tumor growth
and metastasis. Recently, it has been shown that down modulation of
EZH2 in murine models blocks prostate cancer metastasis (Min et
al., "An oncogene-tumor suppressor cascade drives metastatic
prostate cancer by coordinately activating Ras and nuclear
factor-kappaB," Nat Med. 2010 March; 16(3):286-94). EZH2
overexpression is associated with aggressiveness of certain cancers
such as breast cancer (Kleer et al., Proc. Nat. Acad. Sci. USA
100:11606-11611, 2003). Recent studies also suggest that prostate
cancer specific oncogenic fusion gene TMPRSS2-ERG induces
repressive epigenetic programs via direct activation of EZH2 (Yu et
al., "An Integrated Network of Androgen Receptor, Polycomb, and
TMPRSS2-ERG Gene Fusions in Prostate Cancer Progression," Cancer
Cell. 2010 May 18; 17(5):443-454).
[0059] In some embodiments, compounds of the present invention
modulate the activity of one or more enzymes involved in epigenetic
regulation. In some embodiments, compounds of the present invention
modulate the activity of a histone methyl modifying enzyme, or a
mutant thereof. In some embodiments, compounds of the present
invention modulate EZH2 activity. In some embodiments, compounds of
the present invention down-regulate or suppress the activity of
EZH2. In some embodiments, compounds of the present invention are
antagonists of EZH2 activity.
[0060] In some embodiments, compounds and compositions of the
present invention are useful in treating diseases and/or disorders
associated with a histone methyl modifying enzyme. Accordingly, in
some embodiments, the present invention provides a method of
modulating a disease and/or disorder associated with a histone
methyl modifying enzyme. In some embodiments, the present invention
provides a method of treating a subject suffering from a disease
and/or disorder associated with a histone methyl modifying enzyme
comprising the step of administering a compound or composition of
Formula II.
[0061] In some embodiments, compounds and compositions of the
present invention are useful in treating diseases and/or disorders
associated with overexpression of EZH2. In some embodiments, the
present invention provides a method of treating a subject suffering
from a disease and/or disorder associated with overexpression of
EZH2 comprising the step of administering a compound or composition
of Formula II. In some embodiments, the above method additionally
comprises the preliminary step of determining if the subject is
overexpressing EZH2.
[0062] In some embodiments, compounds and compositions of the
present invention are useful in treating diseases and/or disorders
associated with cellular proliferation. In some embodiments,
compounds and compositions of the present invention are useful in
treating diseases and/or disorders associated with misregulation of
cell cycle or DNA repair. In some embodiments, compounds and
compositions of the present invention are useful in treating
cancer. Exemplary types of cancer include breast cancer, prostate
cancer, colon cancer, renal cell carcinoma, glioblastoma multiforme
cancer, bladder cancer, melanoma, bronchial cancer, lymphoma and
liver cancer.
[0063] The study of EZH2 deletions, missense and frameshift
mutations suggest that EZH2 functions as a tumor suppressor in
blood disorders such as myelodysplastic syndromes (MDS) and myeloid
malignancies (Ernst et al., Nat Genet. 2010 August; 42(8):722-6;
Nikoloski et al., Nat Genet. 2010 August; 42(8):665-7).
Accordingly, in some embodiments, compounds and compositions of the
present invention are useful in treating diseases and/or disorders
associated with the presence of a mutant form of EZH2. In some
embodiments, compounds and compositions of the present invention
are useful in treating diseases and/or disorders associated with
the presence of Y641N EZH2. In some embodiment, the disease or
disorder associated with the presence of a mutant form of EZH2 is a
human B cell lymphoma. In some embodiments, the disease and/or
disorder associated with the presence of Y641N EZH2 is follicular
lymphoma or diffuse large-B-cell lymphoma. In some embodiments,
compounds or compositions of the present invention are useful in
treating blood disorders, such as myelodysplastic syndromes,
leukemia, anemia and cytopenia. Sneeringer et al., "Coordinated
activities of wild-type plus mutant EZH2 drive tumor-associated
hypertrimethylation of lysine 27 on histone H3 (H3K27) in human
B-cell lymphomas," Proceedings of the National Academy of Sciences,
PNAS Early Edition published ahead of print on Nov. 15, 2010.
[0064] In some embodiments, the present invention provides a method
of reducing the activity of EZH2 in a subject comprising the step
of administering a compound or composition of Formula II. In some
embodiments, the present invention provides a method of reducing
the activity of wide-type EZH2 in a subject comprising the step of
administering a compound or composition of Formula II. In some
embodiments, the present invention provides a method of reducing
the activity of a mutant form of EZH2 in a subject comprising the
step of administering a compound or composition of Formula II. In
some embodiments, the present invention provides a method of
reducing the activity of a mutant form of EZH2 in a subject
comprising the step of administering a compound or composition of
Formula II, wherein the mutant form of EZH2 is Y641N EZH2. In some
embodiments, the present invention provides a method of treating a
subject suffering from a disease and/or disorder associated with
EZH2 comprising the step of administering a compound or composition
of Formula II. In some embodiments, the present invention provides
a method of treating a subject suffering from a disease and/or
disorder associated with wide-type EZH2 comprising the step of
administering a compound or composition of Formula II. In some
embodiments, the present invention provides a method of treating a
subject suffering from a disease and/or disorder associated with a
mutant form of EZH2 comprising the step of administering a compound
or composition of Formula II. In some embodiments, the present
invention provides a method of treating a subject suffering from a
disease and/or disorder associated with a mutant form of EZH2
comprising the step of administering a compound or composition of
Formula II, wherein the mutant form of EZH2 is Y641N EZH2. In some
embodiments, the above method additionally comprises the
preliminary step of determining if the subject is expressing a
mutant form of EZH2, such as Y641N EZH2. In some embodiments, the
present invention provides a method of reducing the activity of a
mutant form of EZH2, such as Y641N EZH2, in a subject in need
thereof comprising the step of administering a compound or
composition of Formula II. In some embodiments, the present
invention provides a method of treating a subject suffering from a
disease and/or disorder associated with a mutant form of EZH2
comprising the step of administering a compound or composition of
Formula II. In some embodiments, the above method additionally
comprises the preliminary step of determining if the subject is
expressing a mutant form of EZH2, such as Y641N EZH2. In some
embodiments, that determination is made by determining if the
subject has increased levels of histone H3 Lys-27-specific
trimethylation (H3K27me3), as compared to a subject known not to
express a mutant form of EZH2.
Equivalents
[0065] The representative examples that follow are intended to help
illustrate the invention, and are not intended to, nor should they
be construed to, limit the scope of the invention. Indeed, various
modifications of the invention and many further embodiments
thereof, in addition to those shown and described herein, will
become apparent to those skilled in the art from the full contents
of this document, including the examples that follow and the
references to the scientific and patent literature cited herein. It
should further be appreciated that the contents of those cited
references are incorporated herein by reference to help illustrate
the state of the art.
[0066] It will be appreciated that for compound preparations
described herein, when reverse phase HPLC is used to purify a
compound, a compound may exist as an acid addition salt. In some
embodiments, a compound may exist as a formic acid or mono-, di-,
or tri-trifluoroacetic acid salt.
[0067] It will further be appreciated that the present invention
contemplates individual compounds described herein. Where
individual compounds exemplified are isolated and/or characterized
as a salt, for example, as a trifluoroacetic acid salt, the present
invention contemplates a free base of the salt, as well as other
pharmaceutically acceptable salts of the free base.
[0068] The following examples contain important additional
information, exemplification and guidance that can be adapted to
the practice of this invention in its various embodiments and the
equivalents thereof.
[0069] Procedures for preparing the compounds exemplified below, as
well as additional compounds/intermediates in the synthetic schemes
can be found in International Application No. PCT/US2013/025639,
the contents of which are incorporated herein by reference.
EXAMPLES
[0070] As depicted in the Examples below, in certain exemplary
embodiments, compounds are prepared according to the following
general procedures. It will be appreciated that, although the
synthetic methods and Schemes depict the synthesis of certain
compounds of the present invention, the following methods and other
methods known to one of ordinary skill in the art can be applied to
all compounds and subclasses and species of each of these
compounds, as described herein.
[0071] Unless otherwise noted, all solvents, chemicals, and
reagents were obtained commercially and used without purification.
The .sup.1H NMR spectra were obtained in CDCl.sub.3, d.sub.6-DMSO,
CD.sub.3OD, or d.sub.6-acetone at 25.degree. C. at 300 MHz on an
OXFORD (Varian) with chemical shift (.delta., ppm) reported
relative to TMS as an internal standard. HPLC-MS chromatograms and
spectra were obtained with Shimadzu LC-MS-2020 system. Chiral
analysis and purification were obtained with Yilite P270.
Example 1
Synthesis of Compounds 327 and 346 and Related Compounds and
Intermediates
[0072] The title compounds of this Example and other related
compounds were prepared according to the following general scheme.
In addition, where indicated, modifications of this scheme are
disclosed for the synthesis of still additional related compounds
of the invention and intermediates thereof.
##STR00004## ##STR00005##
Step 1: (S,E)-tert-butyl
4-(((tert-butylsulfinyl)imino)methyl)piperidine-1-carboxylate:
(S)-2-methylpropane-2-sulfinamide
##STR00006##
[0073] To a round bottomed flask charged with a magnetic stir bar
was added (S)-2-methylpropane-2-sulfinamide (20.46 g, 169 mmol),
tert-butyl 4-formylpiperidine-1-carboxylate (30 g, 141 mmol), DCM
(300 mL), and Ti(OEt).sub.4 (59.0 ml, 281 mmol). The solution was
stirred at room temperature for 3 h before it was quenched with
brine (80 mL). The solution was stirred for 30 minutes before
filtering. The filter cake was washed with DCM and the filtrate was
placed in a separatory funnel and washed with water. The organics
layer was dried over Na.sub.2SO.sub.4, filtered, and concentrated
in vacuo. The crude residue solidified to the title compound (29 g,
92 mmol, 65.1% yield) m/z 217.
[0074] The intermediate shown in the following table was prepared
according to the general procedure outlined in Step 1 using the
appropriate starting materials and modifications.
TABLE-US-00001 Name Structure m/z (S,E)-2-methyl-N-((tetrahydro-
2H-pyran-4- yl)methylene)propane- 2-sulfinamide ##STR00007##
Step 2: Tert-butyl 4-((S)-1-((R or
S)-1,1-dimethylethylsulfinamido)ethyl)piperidine-1-carboxylate
##STR00008##
[0075] To a round bottomed flask charged with a magnetic stir bar
was added (S,E)-tert-butyl
4-((tert-butylsulfinylimino)methyl)piperidine-1-carboxylate (36.4
g, 115 mmol), DCM (400 mL), and the solution was cooled to
0.degree. C. in an ice bath with stirring. To this solution was
added MeMgBr (77 ml, 230 mmol) (3M in diethyl ether) and the
reaction stirred for 4 h while warming to room temperature. The
reaction was carefully quenched via the addition of saturated
aqueous NH.sub.4Cl. The solid were broken up by the addition of 1N
HCl. The layers were separated and the aqueous phase was extracted
with DCM. The combined organics phase was dried over
Na.sub.2SO.sub.4, filtered, and concentrated in vacuo to afford the
title compound (29 g, >9:1 dr) which is used without further
purification in the next step.
[0076] The intermediate shown in the following table was prepared
according to the general procedure outlined in Step 2 using the
appropriate starting materials and modifications.
TABLE-US-00002 Name Structure m/z (S)-2-methyl-N-((R or S)-1-
(tetrahydro-2H-pyran-4- yl)ethyl)propane-2-sulfinamide ##STR00009##
234
Step 3: (R or S)-tert-butyl
4-(1-aminoethyl)piperidine-1-carboxylate
##STR00010##
[0077] To a 1 L round bottomed flask charged with a magnetic stir
bar was added crude tert-butyl
4-((S)-1-((S)-1,1-dimethylethylsulfinamido)ethyl)piperidine-1-carboxylate
(29 g) was taken up in MeOH (200 mL) before addition of a 4 N
solution of HCl in 1,4-dioxane (24.06 ml, 96 mmol). The resulting
solution was then stirred at room temperature for 1 h at rt. The
methanol was then removed in vacuo to afford viscous oil which was
treated with sat'd aqueous NaHCO.sub.3 (.about.500 mL) and
extracted with ethyl acetate (2.times.500 mL). This organic phase
was combined, dried with MgSO.sub.4, filtered, and solvent was then
removed in vacuo affording the title compound (22 g) which was used
without further purification.
[0078] The intermediate shown in the following table was prepared
according to the general procedure outlined in Step 3 using the
appropriate starting materials.
TABLE-US-00003 Name Structure m/z (R or
S)-1-(tetrahydro-2H-pyran-4-yl)ethanamine ##STR00011## 130
Step 4: Methyl 2-(2-bromophenyl)-3-oxobutanoate
##STR00012##
[0079] A round bottomed flask was charged with a magnetic stir bar
and methyl 2-(2-bromophenyl)acetate (25 g, 109 mmol) and THF (50
mL). This solution was cooled to -78.degree. C. before drop wise
addition of a 1M solution of LiHMDS in THF (218 ml, 218 mmol). The
reaction was stirred for 30 min at -78.degree. C. before addition
of 1-(1H-imidazol-1-yl)ethanone (14.42 g, 131 mmol) dissolved in a
mixture of THF:DMF (112 mL THF, 24 mL DMF). The solution was
stirred for 1 h before quenching with sat'd aqueous NH.sub.4Cl
(.about.250 mL) and diluting with EtOAc. The layers were separated
and the aqueous phase was extracted with EtOAc (.about.2.times.250
mL). The combined organic extract was washed with brine, dried over
Na.sub.2SO.sub.4, filtered, and concentrated in vacuo. The crude
residue was purified via silica gel chromatography using an eluent
of ethyl acetate/hexanes (10:1) to afford methyl
2-(2-bromophenyl)-3-oxobutanoate (32.5 g, 102 mmol, 93% yield).
[0080] The intermediates shown in the following table were prepared
according to the general procedure outlined in Step 4 using the
appropriate starting materials.
TABLE-US-00004 Name Structure m/z methyl 2-(2-bromo-4-
chlorophenyl)-3-oxobutanoate ##STR00013## 304 methyl 2-(2-bromo-4-
methoxyphenyl)-3- oxobutanoate ##STR00014## 302 methyl
2-(2-bromo-4- fluorophenyl)-3-oxobutanoate ##STR00015## 289
Step 5: (R or S, E and Z)-tert-butyl
4-(1-(3-(2-bromophenyl)-4-methoxy-4-oxobut-2-en-2-ylamino)ethyl)piperidin-
e-1-carboxylate
##STR00016##
[0081] To a round bottomed flask was added (R or S)-tert-butyl
4-(1-aminoethyl)piperidine-1-carboxylate (9.35 g, 40.9 mmol), EtOH
(75 mL), and methyl 2-(2-bromophenyl)-3-oxobutanoate (7.40 g, 27.3
mmol) (from Step 4). To this solution was added AcOH (1.563 ml,
27.3 mmol) and the reaction was heated overnight at 85.degree. C.
before cooling to room temperature and concentrating. The crude
residue was purified via silica gel chromatography (330 g, 100%
hexanes to 25% EA in hexanes) to afford the title compound (6.45 g,
13.40 mmol, 49.1% yield).
[0082] The intermediates shown in the following table were prepared
according to the general procedure outlined in Step 5 using the
appropriate starting materials.
TABLE-US-00005 Name Structure m/z (R or S,Z)-methyl 2-(2-
bromophenyl)-3-((1- (tetrahydro-2H-pyran-4- yl)ethyl)amino)but-2-
enoate ##STR00017## 383 (R or S,Z)-methyl 2-(2-
bromo-4-chlorophenyl)-3- ((1-(tetrahydro-2H-pyran-
4-yl)ethyl)amino)but-2- enoate ##STR00018## 417 (R or S,Z)-methyl
2-(2- bromo-4-chlorophenyl)-3- ((1-(tetrahydro-2H-pyran-
4-yl)ethyl)amino)but-2- enoate ##STR00019## 417 (R or S,Z)-methyl
2-(2- bromo-4-fluorophenyl)-3- ((1-(tetrahydro-2H-pyran-
4-yl)ethyl)amino)but-2- enoate ##STR00020## 401
Step 6: (R or S)-methyl
1-(1-(1-(tert-butoxycarbonyl)piperidin-4-yl)ethyl)-2-methyl-1H-indole-3-c-
arboxylate
##STR00021##
[0083] A 250 mL round bottom flask was charged with a magnetic stir
bar, (R or S,Z)-tert-butyl
4-(1-(3-(2-bromophenyl)-4-methoxy-4-oxobut-2-en-2-ylamino)ethyl)piperidin-
e-1-carboxylate (3.33 g, 6.92 mmol), RuPhos Pre-catalyst II
(Methanesulfonato(2-dicyclohexylphosphino-2',6'-di-i-propoxy-1,1'-bipheny-
l)(2-amino-1,1'-biphenyl-2-yl) palladium(II)) (0.463 g, 0.553
mmol), dicyclohexyl(2',6'-diisopropoxybiphenyl-2-yl)phosphine
(0.387 g, 0.830 mmol), anhydrous 1,4-dioxane (27.7 ml, 6.92 mmol),
and sodium methoxide (0.561 g, 10.38 mmol). The reaction mixture
was purged and back-filled with nitrogen and heated to 100.degree.
C. with stirring overnight before being allowed to cool to rt. The
reaction was diluted with ethyl acetate (.about.100 ml) and the
mixture was filtered through a bed of diatomaceous earth. The
filtrate was pre-absorbed onto silica gel (.about.30 g) and
purified via silica gel chromatography (120 g) using ethyl
acetate/hexanes (1:1) as eluent to afford the title compound (2.01
g, 4.77 mmol, 68.9% yield).
[0084] The intermediates shown in the following table were prepared
according to the general procedure outlined in Step 6 using the
appropriate starting materials.
TABLE-US-00006 Name Structure m/z (R or S)-methyl 2-methyl-1-
(1-(tetrahydro-2H-pyran- 4-yl)ethyl)-1H-indole-3- carboxylate
##STR00022## 302 (R or S)-methyl 6-chloro-2-
methyl-1-(1-(tetrahydro- 2H-pyran-4-yl)ethyl)-1H-
indole-3-carboxylate ##STR00023## 337 (R or S)-methyl 6-methoxy-
2-methyl-1-(1-(tetrahydro- 2H-pyran-4-yl)ethyl)-
1H-indole-3-carboxylate ##STR00024## 332 (R or S)-methyl 6-fluoro-
2-methyl-1-(1-(tetrahydro- 2H-pyran-4-yl)ethyl)-1H-
indole-3-carboxylate ##STR00025## 320
Step 7: (R or
S)-2-methyl-1-(1-(tetrahydro-2H-pyran-4-yl)ethyl)-1H-indole-3-carboxylic
acid
##STR00026##
[0085] A 1 L round bottom flask was charged with a magnetic stir
bar, (R or S)-methyl
2-methyl-1-(1-(tetrahydro-2H-pyran-4-yl)ethyl)-1H-indole-3-carboxylate
(11.60 g, 38.5 mmol), ethanol (96 ml, 38.5 mmol), and 6 N aqueous
NaOH (64.1 ml, 385 mmol). The flask was fitted with a reflux
condenser and heated to reflux for 6 h before being allowed to cool
to rt. The volatiles were removed in vacuo and the resulting
mixture was poured into 10% HCl (.about.300 mL). A precipitate
formed which was collected via vacuum filtration using a Buchner
funnel. The filter cake was rinsed with an additional portion of
water (.about.200 mL), collected, and dried under vacuum to afford
the title compound (10.87 g, 35.9 mmol, 93% yield) as an off-white
solid.
[0086] The intermediates shown in the following table were prepared
according to the general procedure outlined in Step 7 using the
appropriate starting materials.
TABLE-US-00007 Name Structure m/z (R or S)-2-methyl-1-(1-
(tetrahydro-2H-pyran- 4-yl)ethyl)-1H-indole- 3-carboxylic acid
##STR00027## 287 (R or S)-6-chloro-2- methyl-1-(1-
(tetrahydro-2H-pyran- 4-yl)ethyl)-1H-indole- 3-carboxylic acid
##STR00028## 321 (R or S)-6-methoxy-2- methyl-1-(1-
(tetrahydro-2H-pyran- 4-yl)ethyl)-1H-indole- 3-carboxylic acid
##STR00029## 317 (R or S)-6-fluoro-2- methyl-1-(1-
(tetrahydro-2H-pyran- 4-yl)ethyl)-1H-indole- 3-carboxylic acid
##STR00030## 306 (R or S)-2-methyl-6- (pyridin-3-yl)-1-(1-
(tetrahydro-2H-pyran- 4-yl)ethyl)-1H-indole- 3-carboxylic acid
##STR00031## 365
Step 8: (R or S)-tert-butyl
4-(1-(3-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methylcarbamoy-
l)-2-methyl-1H-indol-1-yl)ethyl)piperidine-1-carboxylate (Compound
327)
##STR00032##
[0087] A 250 mL round bottom flask was charged with a magnetic stir
bar, (R or
S)-1-(1-(1-(tert-butoxycarbonyl)piperidin-4-yl)ethyl)-2-methyl-1H-i-
ndole-3-carboxylic acid (1.950 g, 5.05 mmol),
3-(aminomethyl)-4-methoxy-6-methylpyridin-2(1H)-one hydrochloride
(2.065 g, 10.09 mmol), DMF (25.2 ml, 5.05 mmol), Hunig's base (3.52
ml, 20.18 mmol). The reaction mixture was cooled to 0.degree. C.
and COMU (2.16 g, 5.05 mmol) was added. The reaction was allowed to
stir overnight to room temperature. The reaction mixture was
diluted with water and extracted with EtOAc. The combined organic
extract was washed with brine, dried with MgSO.sub.4, filtered and
conc. in vacuo to afford the crude material which was purified via
silica gel chromatography (120 g) using MeOH/ethyl acetate (1:5) as
eluent to afford the title compound (1.86 g, 3.29 mmol, 65.3%
yield). LCMS 537 (M+1).sup.+ 1 H NMR (400 MHz, DMSO-d.sub.6)
.delta.=11.83-11.71 (m, 1H), 7.80 (br. s., 1H), 7.73 (d, J=7.6 Hz,
1H), 7.62 (d, J=7.8 Hz, 1H), 7.06 (td, J=7.1, 14.4 Hz, 2H), 6.21
(s, 1H), 4.32 (br. s., 2H), 4.16 (br. s., 1H), 4.02 (br. s., 1H),
3.85 (s, 3H), 3.75 (br. s., 1H), 2.70 (br. s., 1H), 2.58 (s, 3H),
2.37 (br. s., 1H), 2.21 (s, 3H), 1.90 (d, J=12.9 Hz, 1H), 1.53 (d,
J=6.9 Hz, 3H), 1.35 (s, 10H), 1.21 (br. s., 1H), 0.89 (d, J=8.7 Hz,
1H), 0.67 (d, J=11.8 Hz, 1H).
[0088] The compounds shown in the following table were prepared
according to the general procedure outlined in Step 8 using the
appropriate starting materials. The structures of the compounds are
shown in FIG. 1.
TABLE-US-00008 Compound Number Name .sup.1H NMR m/z 435 (R or
S)-tert-butyl 4-(1-(3-(((4,6- 521 dimethyl-2-oxo-1,2-
dihydropyridin-3- yl)methyl)carbamoyl)-2-methyl-
1H-indol-1-yl)ethyl)piperidine-1- carboxylate 436 (R or
S)-tert-butyl 4-(1-(3-(((4- 573 (difluoromethoxy)-6-methyl-2-
oxo-1,2-dihydropyridin-3- yl)methyl)carbamoyl)-2-methyl-
1H-indol-1-yl)ethyl)piperidine-1- carboxylate 437 (R or
S)-tert-butyl 4-(1-(3-(((4- 535 ethyl-6-methyl-2-oxo-1,2-
dihydropyridin-3- yl)methyl)carbamoyl)-2-methyl-
1H-indol-1-yl)ethyl)piperidine-1- carboxylate 298 (R or
S)-N-((4-methoxy-6- (400 MHz, DMSO-d.sub.6) .delta. 11.60 (s, 438
methyl-2-oxo-1,2-dihydropyridin- 1H), 7.73-7.62 (m, 3H), 7.60 (d,
3-yl)methyl)-2-methyl-1-(1- 2H) 7.07-7.05 (m, 2H), 6.15 (s, 1H)
(tetrahydro-2H-pyran-4-yl)ethyl)- 4.33 (s, 1H), 4.21-4.11 (m, 1H),
1H-indole-3-carboxamide 3.92 (br. d., 1H), 3.65 (d, 1H), 3.34- 3.32
(m, 1H), 3.02 (t, 1H), 2.61 (s, 3H), 2.48-2.44 (m, 1H), 2.20 (s,
3H), 1.84-1.81 (m, 1H), 1.54 (d, 3H), 1.40-1.38 (m, 12H), 1.25-1.22
(m, 1H), 1.08-1.04 (m, 1H), 0.86 (br. s., 1H), 0.58 (br. d., 1H)
300 (R or S)-6-fluoro-N-((4-methoxy- (400 MHz, DMSO-d.sub.6)
.delta. = 11.57 456 6-methyl-2-oxo-1,2- (br. s., 1 H), 7.75-7.67
(m, 2 H), dihydropyridin-3-yl)methyl)-2- 7.48 (d, J = 10.7 Hz, 1
H), 6.90 (t, J = methyl-1-(1-(tetrahydro-2H- 8.5 Hz, 1 H), 6.13 (s,
1 H), 4.29 pyran-4-yl)ethyl)-1H-indole-3- (d, J = 4.5 Hz, 2 H),
4.12 (br. s., 1 carboxamide H), 3.94-3.87 (m, 1 H), 3.83 (s, 3 H),
3.64 (dd, J = 3.6, 10.9 Hz, 1 H), 3.35 (br. s., 1 H), 3.05 (br. s.,
1 H), 2.56 (s, 3 H), 2.45-2.37 (m, 1 H), 2.18 (s, 3 H), 1.81 (d, J
= 12.7 Hz, 1 H), 1.50 (d, J = 6.9 Hz, 3 H), 1.40- 1.29 (m, 1 H),
1.11-0.99 (m, 1 H), 0.61 (br. s., 1 H) 314 (R or
S)-6-chloro-N-((4-methoxy- (400 MHz, DMSO-d.sub.6) .delta. 11.57
(s, 1 472 6-methyl-2-oxo-1,2- H), 7.75 (s, 2 H), 7.66 (d, J = 8.9
dihydropyridin-3-yl)methyl)-2- Hz, 1 H), 7.08 (d, J = 8.5 Hz, 1 H),
methyl-1-(1-(tetrahydro-2H- 6.14 (s, 1 H), 4.30 (d, J = 4.5 Hz, 2
pyran-4-yl)ethyl)-1H-indole-3- H), 4.21-4.05 (m, 2 H), 3.91 (d, J =
carboxamide 11.4 Hz, 1 H), 3.85 (s, 3 H), 3.65 (d, J = 10.5 Hz, 1
H), 3.02 (t, J = 11.3 Hz, 1 H), 2.58 (s, 3 H), 2.46- 2.31 (m, 1 H),
2.19 (s, 3 H), 1.82 (d, J = 12.0 Hz, 1 H), 1.59-1.45 (m, 4 H),
1.44-1.29 (m, 1 H), 0.57 (d, J = 12.9 Hz, 1 H) 321 (R or
S)-6-methoxy-N-((4- (400 MHz, DMSO-d.sub.6) .delta. = 11.59 (s, 468
methoxy-6-methyl-2-oxo-1,2- 1 H), 7.67-7.59 (m, 2 H), 7.03 (s,
dihydropyridin-3-yl)methyl)-2- 1 H), 6.75-6.68 (m, 1 H), 6.14 (s,
methyl-1-(1-(tetrahydro-2H- 1 H), 4.30 (d, J = 5.1 Hz, 2 H), 4.10
pyran-4-yl)ethyl)-1H-indole-3- (dd, J = 7.5, 10.4 Hz, 1 H), 3.91
carboxamide (dd, J = 3.0, 11.3 Hz, 1 H), 3.83 (s, 3 H), 3.80-3.76
(m, 3 H), 3.68- 3.60 (m, 1 H), 3.38-3.32 (m, 1 H), 3.10-3.00 (m, 1
H), 2.56 (s, 3 H), 2.19 (s, 3 H), 1.83 (d, J = 12.7 Hz, 1 H),
1.55-1.43 (m, 4 H), 1.34 (br. s., 1 H), 1.10-0.96 (m, 1 H), 0.62
(d, J = 13.4 Hz, 1 H) 335 (R or S)-N-((4- 474
(difluoromethoxy)-6-methyl-2- oxo-1,2-dihydropyridin-3-
yl)methyl)-2-methyl-1-(1- (tetrahydro-2H-pyran-4-yl)ethyl)-
1H-indole-3-carboxamide 394 (R or S)-N-((4,6-dimethyl-2-oxo- 422
1,2-dihydropyridin-3-yl)methyl)- 2-methyl-1-(1-(tetrahydro-2H-
pyran-4-yl)ethyl)-1H-indole-3- carboxamide 442 (R or
S)-6-chloro-N-((4,6- 456 dimethyl-2-oxo-1,2-
dihydropyridin-3-yl)methyl)-2- methyl-1-(1-(tetrahydro-2H-
pyran-4-yl)ethyl)-1H-indole-3- carboxamide
Step 9: (R or
S)--N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methyl-
-1-(1-(piperidin-4-yl)ethyl)-1H-indole-3-carboxamide hydrochloride
(Compound 326)
##STR00033##
[0089] A 250 mL round bottom flask was charged with a magnetic stir
bar, (R or S)-tert-butyl
4-(1-(3-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methylcarbamoy-
l)-2-methyl-1H-indol-1-yl)ethyl)piperidine-1-carboxylate (Compound
327) (1.850 g, 3.45 mmol), MeOH (13.79 ml, 3.45 mmol), and HCl
(2.59 ml, 10.34 mmol) (4 N in dioxane). The reaction was allowed to
stir at rt for 6 h before being conc. in vacuo to afford the title
compound (1.65 g, 3.14 mmol, 91% yield). LCMS 437 (M+1).sup.+.
[0090] The compound shown in the following table was prepared
according to the general procedure outlined in Step 9 using the
appropriate starting materials. The structure of this compound is
shown in FIG. 1.
TABLE-US-00009 Compound Number Name .sup.1H NMR m/z 376 (R or
S)-1-(1-(1- (400 MHz, DMSO-d.sub.6) .delta. 12.27-12.10 (m, 1 476
(azetidin-3-yl)piperidin- H), 11.96-11.72 (m, 1 H), 9.80 (br. s., 1
4-yl)ethyl)-N-((4,6- H), 9.19 (br. s., 2 H), 7.89-7.67 (m, 2 H),
dimethyl-2-oxo-1,2- 7.62 (d, J = 7.6 Hz, 1 H), 7.09 (quin, J = 6.6
Hz, dihydropyridin-3- 2 H), 5.99 (s, 1 H), 4.59-4.36 (m, 3 H),
yl)methyl)-2-methyl-1H- 4.24-3.95 (m, 2 H), 3.48 (d, J = 13.2 Hz, 1
indole-3-carboxamide H), 3.17 (d, J = 12.0 Hz, 1 H), 2.87 (br. s.,
1 hydrochloride H), 2.70 (br. s., 2 H), 2.58 (s, 3 H), 2.34-2.25
(m, 3 H), 2.19-2.10 (m, 3 H), 1.75 (d, J = 12.3 Hz, 1 H), 1.57 (d,
J = 6.7 Hz, 3 H), 1.47 (d, J = 12.7 Hz, 2 H), 1.33-1.21 (m, 2 H),
0.85 (d, J = 13.6 Hz, 1 H)
Step 10: (R or S)-isopropyl
4-(1-(3-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methylcarbamoy-
l)-2-methyl-1H-indol-1-yl)ethyl)piperidine-1-carboxylate (Compound
346)
##STR00034##
[0091] A 250 mL round bottom flask was charged with a magnetic stir
bar, (R or
S)--N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2--
methyl-1-(1-(piperidin-4-yl)ethyl)-1H-indole-3-carboxamide
hydrochloride (0.467 g, 0.987 mmol), DMF (2.468 ml, 0.987 mmol),
THF (2.468 ml, 0.987 mmol), and N-ethyl-N-isopropylpropan-2-amine
(0.638 g, 4.94 mmol). The reaction was cooled to 0.degree. C. and
isopropyl carbonochloridate (0.160 ml, 1.086 mmol) was added drop
wise via syringe. The reaction was allowed to stir for 2 h to rt
and was then treated with 5 N LiOH for 1 h to remove any acylated
pyridone. This material was extracted with ethyl acetate, washed
with brine, dried with MgSO.sub.4 and filtered and conc. in vacuo.
The resulting material was purified via silica gel chromatography
(50 g) using ethyl acetate/MeOH (5:1) as eluent to afford pure
title compound as a pale yellow solid (0.300 g, 0.545 mmol, 55.2%
yield). LCMS 523 (M+1).sup.+; .sup.1H NMR (DMSO-d6, 400 MHz)
.delta. 11.59 (br. s., 1H), 7.74 (d, J=7.8 Hz, 1H), 7.69 (t, J=4.9
Hz, 1H), 7.62 (d, J=7.8 Hz, 1H), 7.13-7.01 (m, 2H), 6.15 (s, 1H),
4.78-4.67 (m, 1H), 4.32 (d, J=4.9 Hz, 2H), 4.23-4.12 (m, 1H),
4.12-4.02 (m, 1H), 3.84 (s, 3H), 3.82-3.74 (m, 1H), 2.79-2.66 (m,
1H), 2.58 (s, 3H), 2.46-2.34 (m, 2H), 2.20 (s, 3H), 1.96-1.88 (m,
1H), 1.58-1.46 (m, 4H), 1.15 (d, J=6.0 Hz, 6H), 0.95-0.89 (m, 1H),
0.74-0.65 (m, 1H).
[0092] The compounds shown in the following table were prepared
according to the general procedure outlined in Step 10 using the
appropriate starting materials. The structures of the compounds are
shown in FIG. 1.
TABLE-US-00010 Compound Number Name .sup.1H NMR m/z 336 (R or
S)-N-((4-methoxy-6-methyl-2- (400 MHz, DMSO-d.sub.6) .delta. =
11.59 (s, 515 oxo-1,2-dihydropyridin-3- 1 H), 7.78-7.66 (m, 2 H),
yl)methyl)-2-methyl-1-(1-(1- 7.64-7.57 (m, 1 H), 7.06 (s, 2
(methylsulfonyl)piperidin-4- H), 6.14 (s, 1 H), 4.31 (d, J = 4.9
Hz, yl)ethyl)-1H-indole-3-carboxamide 2 H), 4.25-4.15 (m, 1 H),
3.83 (s, 3 H), 3.63 (s, 1 H), 3.40-3.33 (m, 1 H), 2.79 (s, 3 H),
2.75-2.65 (m, 1 H), 2.60 (s, 3 H), 2.45-2.27 (m, 1 H), 2.19 (s, 3
H), 2.06-1.98 (m, 1 H), 1.55 (d, J = 6.9 Hz, 3 H), 1.45-1.36 (m, 1
H), 1.28-1.18 (m, 1 H), 1.14-1.03 (m, 1 H), 0.83-0.74 (m, 1 H) 337
(R or S)-1-(1-(1-(2-hydroxy-2- (400 MHz, DMSO-d.sub.6) .delta. 523
methylpropanoyl)piperidin-4- 11.58 (br. s., 1 H), 7.77-7.67 (m, 2
yl)ethyl)-N-((4-methoxy-6-methyl-2- H), 7.66-7.60 (m, 1 H), 7.06
(s, oxo-1,2-dihydropyridin-3- 2 H), 6.14 (s, 1 H),
yl)methyl)-2-methyl-1H-indole-3- 5.32-5.23 (m, 1 H), 4.31 (d, J =
4.5 Hz, 2 carboxamide H), 4.19-4.10 (m, 1 H), 3.83 (s, 3 H),
2.75-2.62 (m, 2 H), 2.58 (s, 3 H), 2.19 (s, 4 H), 2.00-1.90 (m, 2
H), 1.54 (d, J = 6.7 Hz, 3 H), 1.32-1.18 (m, 8 H), 0.87-0.78 (m, 1
H), 0.77-0.67 (m, 1 H) 342 (R or S)-1-(1-(1-isobutyrylpiperidin-
(400 MHz, DMSO-d.sub.6) .delta. = 11.59 (s, 507
4-yl)ethyl)-N-((4-methoxy-6-methyl- 1 H), 7.75 (d, J = 7.4 Hz, 1
2-oxo-1,2-dihydropyridin-3- H), 7.72-7.67 (m, 1 H),
yl)methyl)-2-methyl-1H-indole-3- 7.64 (d, J = 8.0 Hz, 1 H),
carboxamide 7.14-7.01 (m, 2 H), 6.15 (s, 1 H), 4.58-4.46 (m, 1 H),
4.32 (d, J = 4.9 Hz, 2 H), 4.09-3.99 (m, 1 H), 3.84 (s, 3 H),
3.81-3.72 (m, 1 H), 3.08-2.97 (m, 1 H), 2.92-2.81 (m, 1 H),
2.78-2.65 (m, 3 H), 2.59 (br. s., 3 H), 2.20 (s, 3 H), 2.03-1.90
(m, 1 H), 1.59-1.47 (m, 4 H), 1.02-0.86 (m, 6 H), 0.78-0.69 (m, 1
H) 344 (R or S)-N-((4-(difluoromethoxy)-6- (400 MHz, DMSO-d.sub.6)
.delta. 551 methyl-2-oxo-1,2-dihydropyridin-3- 12.02-11.95 (m, 1
H), 7.74 (d, J = 8.0 Hz, yl)methyl)-2-methyl-1-(1-(1- 1 H),
7.66-7.57 (m, 2 H), (methylsulfonyl)piperidin-4- 7.11-7.00 (m, 2
H), 6.08 (s, 1 yl)ethyl)-1H-indole-3-carboxamide H), 4.32 (d, J =
4.5 Hz, 2 H), 4.18 (d, J = 7.1 Hz, 1 H), 3.64 (d, J = 12.3 Hz, 1
H), 3.36 (d, J = 12.0 Hz, 1 H), 2.79 (s, 3 H), 2.75-2.65 (m, 2 H),
2.58 (s, 3 H), 2.45-2.27 (m, 2 H), 2.20 (s, 3 H), 2.07-1.98 (m, 1
H), 1.55 (d, J = 6.9 Hz, 3 H), 1.40 (d, J = 8.2 Hz, 1 H), 1.10 (d,
J = 8.9 Hz, 1 H), 0.79 (d, J = 12.5 Hz, 1 H) 345 (R or S)-1-(1-(1-
(400 MHz, DMSO-d.sub.6) .delta. 529
(ethylsulfonyl)piperidin-4-yl)ethyl)- 11.57 (s, 1 H), 7.75 (d, J =
8.0 Hz, 1 N-((4-methoxy-6-methyl-2-oxo-1,2- H), 7.69 (t, J = 5.0
Hz, 1 H), dihydropyridin-3-yl)methyl)-2- 7.62 (d, J = 7.4 Hz, 1 H),
methyl-1H-indole-3-carboxamide 7.06 (d, J = 7.1 Hz, 2 H), 6.15 (s,
1 H), 4.32 (d, J = 5.1 Hz, 2 H), 4.25-4.15 (m, 1 H), 3.84 (s, 3 H),
3.73-3.65 (m, 1 H), 3.45-3.36 (m, 1 H), 3.02-2.93 (m, J = 7.8 Hz, 2
H), 2.87-2.77 (m, 1 H), 2.75-2.66 (m, 1 H), 2.60 (s, 3 H),
2.42-2.30 (m, 1 H), 2.20 (s, 3 H), 2.06-1.97 (m, 1 H), 1.58-1.48
(m, 4 H), 1.42-1.31 (m, 1 H), 1.17 (t, J = 7.5 Hz, 3 H), 1.13-1.00
(m, 1 H), 0.83-0.73 (m, 1 H) 355 (R or S)-1-(1-(4- (400 MHz,
DMSO-d.sub.6) .delta. 543 (isopropylsulfonyl)cyclohexyl)ethyl)-
11.59 (s, 1H), 7.76-7.69 (m, 2H), N-((4-methoxy-6-methyl-2-oxo-1,2-
7.62 (d, 1H), 7.10-7.03 (m, 2H), dihydropyridin-3-yl)methyl)-2-
6.15 (s, 1H), 4.32 (d, 2H), methyl-1H-indole-3-carboxamide
4.29-4.26 (m, 2H), 3.84 (s, 3H), 3.72 (br. d., 1H), 3.45 (br. d.,
1H), 3.26 (tt, 1H), 2.91 (dt, 1H), 2.60 (s, 3H), 2.20 (s, 3H), 1.97
(br. d., 1H), 1.54 (d, 3H), 1.35-1.24 (m, 2H), 1.18 (d, 3H), 1.16
(d, 3H), 1.05-0.78 (m, 2H) 357 (R or S)-isobutyl 4-(1-(3-(((4- (400
MHz, DMSO-d.sub.6) .delta. 537 methoxy-6-methyl-2-oxo-1,2- 11.60
(br.s., 1H), 7.75-7.60 (m, dihydropyridin-3- 3H), 7.10-7.03 (m,
2H), 6.15 (s, yl)methyl)carbamoyl)-2-methyl-1H- 1H) 4.33 (d, 1H),
4.13-4.06 (m, indol-1-yl)ethyl)piperidine-1- 1H), 3.84 (s, 3H),
3.74 (d, 1H), carboxylate 2.80-2.60 (m, 3H), 2.58 (s, 1H),
2.50-2.42 (m, 2H), 1.96-1.90 (m, 1H), 1.54 (d, 3H), 1.25-1.22 (m,
1H), 0.98-0.72 (m, 6H) 368 (R or S)-N-((4,6-dimethyl-2-oxo-1,2-
(400 MHz, DMSO-d.sub.6) .delta. 513
dihydropyridin-3-yl)methyl)-1-(1-(1- 11.59 (s, 1 H), 7.78-7.71 (m,
1 H), (ethylsulfonyl)piperidin-4-yl)ethyl)- 7.66-7.57 (m, 2 H),
7.07 (s, 2 2-methyl-1H-indole-3-carboxamide H), 5.89 (s, 1 H), 4.32
(s, 2 H), 4.25-4.15 (m, 1 H), 3.65-3.59 (m, 1 H), 3.19-3.10 (m, 1
H), 2.98 (d, J = 7.4 Hz, 2 H), 2.87-2.77 (m, 1 H), 2.72-2.65 (m, 1
H), 2.58 (s, 3 H), 2.27 (s, 3 H), 2.12 (s, 3 H), 1.55 (d, J = 6.9
Hz, 4 H), 1.42-1.33 (m, 2 H), 1.17 (t, J = 7.4 Hz, 3 H), 1.12-1.00
(m, 1 H), 0.84-0.74 (m, 1 H) 382 (R or
S)-N-((4,6-dimethyl-2-oxo-1,2- (400 MHz, DMSO-d.sub.6) .delta. 499
dihydropyridin-3-yl)methyl)-2- 11.60 (s, 1 H), 7.75 (d, J = 7.1 Hz,
1 methyl-1-(1-(1- H), 7.65-7.58 (m, 2 H),
(methylsulfonyl)piperidin-4- 7.12-7.02 (m, 2 H), 5.89 (s, 1 H),
yl)ethyl)-1H-indole-3-carboxamide 4.38-4.25 (m, 2 H), 4.20 (dd, J =
7.0, 10.6 Hz, 1 H), 2.80 (s, 3 H), 2.76-2.67 (m, 2 H), 2.59 (s, 3
H), 2.46-2.31 (m, 2 H), 2.27 (s, 3 H), 2.12 (s, 3 H), 1.55 (d, J =
6.9 Hz, 3 H), 1.51 (br. s., 1 H), 1.47-1.34 (m, 1 H), 1.29-1.21 (m,
1 H), 1.17-1.04 (m, 1 H), 0.80 (d, J = 12.9 Hz, 1 H)
Example 2
Synthesis of (R or
S)-1-(1-(1-isopropylpiperidin-4-yl)ethyl)-N-((4-methoxy-6-methyl-2-oxo-1,-
2-dihydropyridin-3-yl)methyl)-2-methyl-1H-indole-3-carboxamide
(Compound 358)
##STR00035##
[0093] A 25 mL vial was charged with a magnetic stir bar, (R or
S)--N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methyl-
-1-(1-(piperidin-4-yl)ethyl)-1H-indole-3-carboxamide hydrochloride,
THF (2.114 ml, 0.211 mmol), propan-2-one (0.061 g, 1.057 mmol), and
sodium triacetoxyborohydride (0.224 g, 1.057 mmol). The reaction
was allowed to stir at rt for 12 h. The reaction was inverse
quenched onto sat'd aqueous NaHCO.sub.3, extracted with ethyl
acetate and conc. in vacuo. The resulting material was treated with
10 mL 7 N ammonia in MeOH and was conc in vacuo to yield material
which was purified via silica gel chromatography (10 g) using
DCM/MeOH/NH.sub.4OH (90:1:0.1) as eluent to afford 33 mg, (0.065
mmol, 31.0% yield) of the title compound as a white solid.). LCMS
479 (M+1).sup.+; .sup.1H NMR (DMSO-d6, 400 MHz)
[0094] .delta. 11.59 (s., 1H), 7.64-7.82 (m, 2H), 7.59 (d, 1H),
6.95-7.17 (m, 2H), 6.15 (s, 1H), 4.32 (d, 2H), 4.04-4.24 (m, 1H),
3.84 (.quadrature., 3H), 2.77-2.93 (.quadrature., 2H), 2.68
(.quadrature., 1H), 2.60 (.quadrature., 3H), 2.20 (.quadrature.,
3H), 2.08-2.15 (.quadrature., 1H), 1.92 (.quadrature., 1H), 1.83
(.quadrature.p. .quadrature.., 1H), 1.54 (.quadrature., 3H),
1.27-1.43 (.quadrature., 2H), 0.91 (.quadrature., 6H), 0.71-0.67
(.quadrature., 2H).
[0095] The compounds shown in the following table were prepared
according to the general procedure outlined in this Example using
the appropriate starting materials. The structures of the compounds
are shown in FIG. 1.
TABLE-US-00011 Compound Number Name .sup.1H NMR m/z 341 (R or
S)-N-((4-methoxy- (400 MHz, DMSO-d.sub.6) .delta. 11.58 (s, 1 H),
493 6-methyl-2-oxo-1,2- 7.76-7.65 (m, 2 H), 7.59 (d, J = 7.8 Hz, 1
dihydropyridin-3- H), 7.10-6.99 (m, 2 H), 6.14 (s, 1 H),
yl)methyl)-2-methyl-1- 4.49 (t, J = 6.4 Hz, 1 H), 4.43 (t, J = 6.5
Hz, 1 (1-(1-(oxetan-3- H), 4.37 (t, J = 6.1 Hz, 1 H), 4.34-4.28 (m,
yl)piperidin-4-yl)ethyl)- 3 H), 4.21-4.10 (m, 1 H), 3.83 (s, 3 H),
1H-indole-3- 3.30-3.23 (m, 1 H), 2.75 (br. s., 1 H), carboxamide
2.71-2.64 (m, 1 H), 2.60 (s, 3 H), 2.19 (s, 4 H), 1.90 (br. s., 1
H), 1.75 (br. s., 1 H), 1.53 (d, J = 6.9 Hz, 3 H), 1.42 (br. s., 2
H), 1.11-0.98 (m, 1 H), 0.72-0.63 (m, 1 H) 343 (R or
S)-N-((4-methoxy- (400 MHz, DMSO-d.sub.6) .delta. 11.58 (s, 1 H),
451 6-methyl-2-oxo-1,2- 7.76-7.65 (m, 2 H), 7.59 (d, J = 7.6 Hz, 1
dihydropyridin-3- H), 7.11-6.99 (m, 2 H), 6.14 (s, 1 H),
yl)methyl)-2-methyl-1- 4.31 (d, J = 5.1 Hz, 2 H), 4.13 (br. s., 1
H), (1-(1-methylpiperidin-4- 3.83 (s, 3 H), 2.83 (d, J = 10.0 Hz, 1
H), yl)ethyl)-1H-indole-3- 2.61-2.52 (m, 5 H), 2.19 (s, 3 H), 2.09
(s, 4 H), carboxamide 1.88 (d, J = 10.7 Hz, 2 H), 1.53 (d, J = 6.7
Hz, 3 H), 1.34 (br. s., 1 H), 1.02 (d, J = 8.2 Hz, 1 H), 0.66 (br.
s., 1 H) 359 (R or S)-N-((4-methoxy- (400 MHz, DMSO-d.sub.6)
.delta. 11.59 (s, 1 H), 495 6-methyl-2-oxo-1,2- 7.77-7.66 (m, 2 H),
7.60 (d, J = 7.8 Hz, 1 dihydropyridin-3- H), 7.12-7.01 (m, 2 H),
6.15 (s, 1 H), yl)methyl)-1-(1-(1-(2- 4.32 (d, J = 4.9 Hz, 2 H),
4.13 (d, J = 7.1 Hz, 1 methoxyethyl)piperidin- H), 3.85 (s, 3 H),
3.36 (t, J = 5.9 Hz, 2 H), 4-yl)ethyl)-2-methyl-1H- 3.19 (s, 3 H),
2.94 (d, J = 10.5 Hz, 1 H), indole-3-carboxamide 2.71-2.56 (m, 5
H), 2.43-2.32 (m, 2 H), 2.24-2.12 (m, 4 H), 1.54 (d, J = 6.9 Hz, 4
H), 1.39-1.27 (m, 2 H), 1.02 (d, J = 8.7 Hz, 1 H), 0.65 (d, J =
12.7 Hz, 1 H) 360 (R or S)-1-(1-(1- (400 MHz, DMSO-d.sub.6) .delta.
11.79-11.45 (m, 1 465 ethylpiperidin-4- H), 7.78-7.65 (m, 2 H),
7.59 (d, J = 7.8 Hz, yl)ethyl)-N-((4- 1 H), 7.14-6.99 (m, 2 H),
6.15 (s, 1 H), methoxy-6-methyl-2- 4.32 (d, J = 4.9 Hz, 2 H),
4.20-4.08 (m, 1 oxo-1,2-dihydropyridin- H), 3.84 (s, 3 H),
2.98-2.89 (m, 1 H), 3-yl)methyl)-2-methyl- 2.71-2.61 (m, 2 H), 2.59
(s, 3 H), 1H-indole-3- 2.27-2.21 (m, 2 H), 2.20 (s, 3 H), 1.94-1.80
(m, 2 H), carboxamide 1.54 (s, 4 H), 1.38-1.28 (m, 1 H), 1.06-0.98
(m, 1 H), 0.93 (t, J = 7.1 Hz, 3 H), 0.71-0.63 (m, 1 H) 363 (R or
S)-ethyl 2-(4-(1-(3- (400 MHz, DMSO-d.sub.6) .delta. 11.59 (br. s.,
1 H), 523 (((4-methoxy-6-methyl- 7.81-7.65 (m, 2 H), 7.60 (d, J =
7.4 Hz, 1 2-oxo-1,2- H), 7.16-6.98 (m, 2 H), 6.15 (s, 1 H),
dihydropyridin-3- 4.32 (d, J = 4.9 Hz, 2 H), 4.23-4.11 (m, 1 H),
yl)methyl)carbamoyl)-2- 4.04 (q, J = 7.0 Hz, 2 H), 3.84 (s, 3 H),
methyl-1H-indol-1- 2.95-2.86 (m, 1 H), 2.60 (s, 5 H), 2.20 (s, 4
H), yl)ethyl)piperidin-1- 1.94-1.79 (m, 2 H), 1.54 (d, J = 6.9 Hz,
4 yl)acetate H), 1.41-1.32 (m, 1 H), 1.15 (t, J = 7.1 Hz, 3 H),
1.04 (d, J = 6.0 Hz, 2 H), 0.71-0.61 (m, 1 H) 366 (R or
S)-N-((4-ethyl-6- (400 MHz, DMSO-d.sub.6) .delta. 11.63 (s, 1 H),
449 methyl-2-oxo-1,2- 7.74 (d, J = 7.6 Hz, 1 H), 7.65-7.56 (m, 2
dihydropyridin-3- H), 7.12-7.01 (m, 2 H), 5.94 (s, 1 H),
yl)methyl)-2-methyl-1- 4.34 (t, J = 5.1 Hz, 2 H), 4.19-4.09 (m, 1
H), (1-(1-methylpiperidin-4- 2.88 (br. s., 1 H), 2.71-2.56 (m, 6
H), yl)ethyl)-1H-indole-3- 2.14 (s, 7 H), 1.91 (d, J = 12.5 Hz, 1
H), 1.54 (d, carboxamide J = 6.9 Hz, 4 H), 1.41-1.31 (m, 2 H), 1.14
(t, J = 7.6 Hz, 3 H), 1.05 (d, J = 9.1 Hz, 1 H), 0.68 (d, J = 12.7
Hz, 1 H) 367 (R or S)-N-((4,6- (400 MHz, DMSO-d.sub.6) .delta.
11.59 (s, 1 H), 435 dimethyl-2-oxo-1,2- 7.74 (d, J = 6.9 Hz, 1 H),
7.65-7.56 (m, 2 dihydropyridin-3- H), 7.12-7.01 (m, 2 H), 5.89 (s,
1 H), yl)methyl)-2-methyl-1- 4.38-4.25 (m, 2 H), 4.20-4.09 (m, 1
H), (1-(1-methylpiperidin-4- 2.95 (br. s., 1 H), 2.68 (br. s., 2
H), 2.58 (s, 3 H), yl)ethyl)-1H-indole-3- 2.27 (s, 3 H), 2.21 (br.
s., 3 H), 2.12 (s, 3 carboxamide H), 1.94 (d, J = 13.8 Hz, 1 H),
1.54 (d, J = 6.9 Hz, 4 H), 1.44-1.31 (m, 2 H), 1.07 (d, J = 12.5
Hz, 1 H), 0.71 (d, J = 13.2 Hz, 1 H) 375 (R or S)-N-((4,6- (400
MHz, DMSO-d.sub.6) .delta. 11.59 (br. s., 1 H), 477
dimethyl-2-oxo-1,2- 7.73 (d, J = 7.6 Hz, 1 H), 7.65-7.55 (m, 2
dihydropyridin-3- H), 7.12-7.00 (m, 2 H), 5.89 (s, 1 H),
yl)methyl)-2-methyl-1- 4.53-4.48 (m, 1 H), 4.47-4.42 (m, 1 H),
(1-(1-(oxetan-3- 4.38 (s, 1 H), 4.31 (t, J = 5.2 Hz, 3 H),
yl)piperidin-4-yl)ethyl)- 4.21-4.10 (m, 1 H), 3.31-3.24 (m, 2 H),
1H-indole-3- 2.81-2.64 (m, 2 H), 2.59 (s, 3 H), 2.26 (s, 3 H),
carboxamide 2.23-2.16 (m, 1 H), 2.12 (s, 3 H), 1.98-1.85 (m, 1 H),
1.81-1.70 (m, 1 H), 1.54 (d, J = 6.9 Hz, 3 H), 1.51-1.22 (m, 1 H),
1.12-0.96 (m, 2 H), 0.73-0.64 (m, 1 H) 380 (R or S)-N-((4-ethyl-6-
(400 MHz, DMSO-d.sub.6) .delta. 11.63 (br. s., 1 H), 491
methyl-2-oxo-1,2- 7.74 (d, J = 7.36 Hz, 1 H), 7.60 (d, J = 8.47 Hz,
dihydropyridin-3- 2 H), 7.06 (quin, J = 7.13 Hz, 3 H),
yl)methyl)-2-methyl-1- 5.94 (s, 1 H), 4.51 (t, J = 6.47 Hz, 1 H),
(1-(1-(oxetan-3- 4.46 (t, J = 6.35 Hz, 1 H), 4.40 (t, J = 6.13 Hz,
yl)piperidin-4-yl)ethyl)- 1 H), 4.37-4.30 (m, 2 H), 1H-indole-3-
4.28-4.11 (m, 1 H), 3.57 (s, 1 H), 3.34 (br. s., 2 H), carboxamide
2.81 (d, J = 10.70 Hz, 1 H), 2.67 (d, J = 14.94 Hz, 1 H), 2.64-2.57
(m, 4 H), 2.21 (d, J = 10.93 Hz, 1 H), 2.14 (s, 3 H), 1.93 (d, J =
12.49 Hz, 1 H), 1.83 (t, J = 11.37 Hz, 1 H), 1.54 (d, J = 6.91 Hz,
3 H), 1.37 (d, J = 10.48 Hz, 1 H), 1.25 (q, J = 6.91 Hz, 1 H), 1.14
(t, J = 7.58 Hz, 3 H), 1.06 (d, J = 9.81 Hz, 1 H), 0.70 (d, J =
12.49 Hz, 1 H) 381 (R or S)-N-((4,6- .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. 505 dimethyl-2-oxo-1,2- 11.60 (br. s., 1 H),
7.74 (d, J = 7.13 Hz, 1 H), dihydropyridin-3- 7.66-7.50 (m, 2 H),
7.15-6.99 (m, 2 H), yl)methyl)-2-methyl-1- 5.89 (s, 1 H), 4.40-4.24
(m, 2 H), (1-(1-(tetrahydro-2H- 4.21-4.07 (m, 1 H), 3.95-3.78 (m, 2
H), 3.57 (s, 1 H), pyran-4-yl)piperidin-4- 3.32-3.17 (m, 3 H), 2.68
(br. s., 1 H), yl)ethyl)-1H-indole-3- 2.58 (s, 3 H), 2.33 (br. s.,
2 H), 2.27 (s, 3 H), carboxamide 2.12 (s, 3 H), 2.00-1.88 (m, 2 H),
1.75 (d, J = 12.04 Hz, 1 H), 1.62 (br. s., 2 H), 1.54 (d, J = 6.91
Hz, 3 H), 1.46-1.30 (m, 2 H), 1.01 (br. s., 1 H), 0.72 (br. s., 1
H) 440 (R or S)-tert-butyl 3-(4- 576 (1-(3-(((4,6-dimethyl-2-
oxo-1,2-dihydropyridin- 3-yl)methyl)carbamoyl)-
2-methyl-1H-indol-1- yl)ethyl)piperidin-1- yl)azetidine-1-
carboxylate 377 (R or S)-N-((4,6- (400 MHz, DMSO-d.sub.6) .delta.
11.63-11.56 (m, 1 490 dimethyl-2-oxo-1,2- H), 7.76-7.70 (m, 1 H),
7.64-7.55 (m, 2 dihydropyridin-3- H), 7.05 (s, 2 H), 5.89 (s, 1 H),
4.56 (s, 4 yl)methyl)-2-methyl-1- H), 4.31 (s, 2 H), 4.19-4.09 (m,
1 H), (1-(1-(1-methylazetidin- 3.36 (d, J = 4.9 Hz, 1 H), 2.77-2.56
(m, 5 H), 3-yl)piperidin-4- 2.26 (s, 3 H), 2.18 (s, 3 H), 2.12 (s,
3 H), yl)ethyl)-1H-indole-3- 1.94-1.85 (m, 1 H), 1.78-1.67 (m, 1
H), carboxamide 1.53 (d, J = 6.9 Hz, 3 H), 1.50-1.45 (m, 1 H),
1.44-1.22 (m, 2 H), 1.07-0.93 (m, 1 H), 0.71-0.61 (m, 1 H)
Example 3
Synthesis of (R or
S)-1-(1-(1-(2-fluoroethyl)piperidin-4-yl)ethyl)-N-((4-methoxy-6-methyl-2--
oxo-1,2-dihydropyridin-3-yl)methyl)-2-methyl-1H-indole-3-carboxamide
(Compound 356)
##STR00036##
[0096] A 25 mL vial was charged with a magnetic stir bar, (R or
S)--N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methyl-
-1-(1-(piperidin-4-yl)ethyl)-1H-indole-3-carboxamide hydrochloride
(0.062 g, 0.131 mmol), K.sub.2CO.sub.3 (0.072 g, 0.524 mmol), MeCN
(0.655 ml, 0.131 mmol), DMF (0.262 ml, 0.131 mmol) and
1-bromo-2-fluoroethane (0.020 ml, 0.262 mmol). The reaction was
capped and heated to 82.degree. C. with stirring for 4 h. The
reaction was allowed to cool to rt, filtered, and the filtrate was
pre-absorbed onto silica gel (12 g). The material was purified via
SiO.sub.2 chromatography (25 g) using DCM/MeOH/Et.sub.3N
(85:15:0.5) as eluent to afford the title compound as an off white
solid (30 mg, 0.059 mmol, 45.1% yield). LCMS 483 (M+1).sup.+;
.sup.1H NMR (DMSO-d6, 400 MHz) .delta. 11.59 (s, 1H), 7.75-7.68 (m,
2H), 7.60 (d, 1H) 7.09-7.03 (m, 2H), 6.15 (s, 1H) 4.53-4.51 (m,
1H), 4.42-4.39 (m, 1H), 4.32 (d, 2H), 4.24-4.2 (m, 1H), 3.84 (s,
3H), 2.98 (br. d., 1H), 2.70-2.49 (m, 4H), 2.60 (s, 3H), 2.20 (s,
3H), 2.01 (dt, 1H), 1.92-1.90 (m, 1H), 1.75-1.71 (m, 1H), 1.54 (d,
3H), 1.38-1.36 (m, 1H), 1.02-0.98 (m, 1H), 0.7-0.66 (br. d.,
1H).
[0097] The compounds shown in the following table were prepared
according to the general procedure outlined in this Example using
the appropriate starting materials. The structures of the compounds
are shown in FIG. 1.
TABLE-US-00012 Compound Number Name .sup.1H NMR m/z 362 (R or
S)-1-(1-(1-(2,2- (400 MHz, DMSO-d.sub.6) .delta. J = 11.60 (br. s.,
1 501 difluoroethyl)piperidin- H), 7.77-7.66 (m, 2 H), 7.60 (d, J =
7.6 Hz, 4-yl)ethyl)-N-((4- 1 H), 7.14-7.00 (m, 2 H), 6.15 (s, 1 H),
methoxy-6-methyl-2- 6.06 (t, J = 55.7 Hz, 1 H), 4.32 (d, J = 4.9
Hz, oxo-1,2-dihydropyridin- 2 H), 4.15 (br. s., 1 H), 3.84 (s, 3
H), 3-yl)methyl)-2-methyl- 3.03-2.93 (m, 2 H), 2.73-2.62 (m, 3 H),
1H-indole-3- 2.60 (s, 3 H), 2.26-2.10 (m, 4 H), carboxamide
1.93-1.79 (m, 1 H), 1.59-1.46 (m, 4 H), 1.41-1.29 (m, 1 H),
1.11-0.97 (m, 1 H), 0.67 (br. s., 1 H) 378 (R or S)-N-((4-methoxy-
(400 MHz, DMSO-d.sub.6) .delta. J = 11.60 (br. s., 1 H), 533
6-methyl-2-oxo-1,2- 7.78-7.66 (m, 2 H), 7.60 (d, J = 8.2 Hz, 1
dihydropyridin-3- H), 7.13-7.00 (m, 2 H), 6.15 (s, 1 H),
yl)methyl)-2-methyl-1- 4.32 (d, J = 4.9 Hz, 2 H), 4.22-4.09 (m, 1
H), (1-(1-(3,3,3- 3.84 (s, 3 H), 3.03-2.91 (m, 1 H),
trifluoropropyl)piperidin- 2.73-2.64 (m, 1 H), 2.60 (s, 3 H),
2.48-2.31 (m, 4-yl)ethyl)-1H-indole-3- 5 H), 2.20 (s, 3 H),
2.01-1.85 (m, 2 H), carboxamide 1.58-1.46 (m, 4 H), 1.36-1.29 (m, 1
H), 1.08-0.98 (m, 1 H), 0.73-0.62 (m, 1 H) 365 (R or
S)-N-((4-methoxy- (500 MHz, DMSO-d.sub.6) .delta. J = 11.59 (s, 1
H), 519 6-methyl-2-oxo-1,2- 7.74 (d, J = 7.6 Hz, 1 H), 7.71-7.66
(m, 1 dihydropyridin-3- H), 7.61 (d, J = 7.8 Hz, 1 H), 7.13-7.01
(m, yl)methyl)-2-methyl-1- 2 H), 6.15 (s, 1 H), 4.32 (d, J = 4.9
Hz, 2 H), (1-(1-(2,2,2- 4.22-4.12 (m, 1 H), 3.84 (s, 3 H),
trifluoroethyl)piperidin- 3.15-2.95 (m, 3 H), 2.75-2.66 (m, 1 H),
2.60 (s, 4-yl)ethyl)-1H-indole-3- 3 H), 2.39-2.31 (m, 1 H), 2.20
(s, 3 H), carboxamide 2.05-1.98 (m, 1 H), 1.92-1.84 (m, 1 H),
1.56-1.46 (m, 4 H), 1.42-1.32 (m, 1 H), 1.11-1.01 (m, 1 H),
0.69-0.62 (m, 1 H) 441 (R or S)-1-(1-(1-(2,2- (400 MHz,
DMSO-d.sub.6) .delta. = 11.59 (s, 1 H), 485
difluoroethyl)piperidin- 7.73 (d, J = 7.8 Hz, 1 H), 7.65-7.55 (m, 2
4-yl)ethyl)-N-((4,6- H), 7.12-7.00 (m, 2 H), 6.22-5.90 (m, 1
dimethyl-2-oxo-1,2- H), 5.89 (s, 1 H), 4.36-4.25 (m, 2 H),
dihydropyridin-3- 4.20-4.09 (m, 1 H), 3.01-2.93 (m, 1 H),
yl)methyl)-2-methyl-1H- 2.72-2.59 (m, 3 H), 2.58 (s, 3 H), 2.26 (s,
3 H), indole-3-carboxamide 2.21-2.13 (m, 2 H), 2.12 (s, 3 H),
1.92-1.79 (m, 2 H), 1.53 (s, 4 H), 1.41-1.29 (m, 1 H), 1.10-0.97
(m, 1 H), 0.70-0.59 (m, 1 H)
Example 4
Synthesis of (R or
S)--N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methyl-
-1-(1-(1-(pyrimidin-2-yl)piperidin-4-yl)ethyl)-1H-indole-3-carboxamide
(Compound 361)
##STR00037##
[0098] To a re-sealable vial was added 2-chloropyrimidine (185 mg,
1.611 mmol), (R or
S)--N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methyl-
-1-(1-(piperidin-4-yl)ethyl)-1H-indole-3-carboxamide hydrochloride
(508 mg, 1.074 mmol), and EtOH (8 mL). To this solution was added
Et.sub.3N (449 .mu.l, 3.22 mmol). The vial was sealed and heated to
100.degree. C. overnight. The solution was allowed to cool to room
temperature and concentrated in vacuo. The crude residue was
purified via silica gel chromatography (hexanes: (3:2 DCM:IPA)) to
afford the title compound as a solid (357 mg, 0.694 mmol, 64.6%
yield). LCMS 515 (M+1).sup.+; .sup.1H NMR (DMSO-d6, 400 MHz)
.delta. 11.60 (s, 1H), 8.30 (d, J=4.7 Hz, 2H), 7.76 (d, J=7.6 Hz,
1H), 7.73-7.64 (m, 2H), 7.14-7.01 (m, 2H), 6.55 (t, J=4.7 Hz, 1H),
6.15 (s, 1H), 4.84-4.75 (m, 1H), 4.57-4.47 (m, 1H), 4.33 (d, J=4.2
Hz, 2H), 4.22-4.11 (m, 1H), 3.84 (s, 3H), 2.92-2.81 (m, 1H),
2.63-2.52 (m, 4H), 2.20 (s, 3H), 2.05-1.94 (m, 1H), 1.61-1.49 (m,
4H), 1.34-1.21 (m, 1H), 1.04-0.91 (m, 1H), 0.83-0.75 (m, 1H).
[0099] The compound shown in the following table was prepared
according to the general procedure outlined in this Example using
the appropriate starting materials. The structure of the compound
is shown in FIG. 1.
TABLE-US-00013 Compound Number Name .sup.1H NMR m/z 373 (R or
S)-N-((4- (400 MHz, DMSO-d.sub.6) .delta. 11.60 (br. s., 1 H), 514
methoxy-6-methyl- 8.06 (d, J = 3.6 Hz, 1 H), 7.82-7.62 (m, 3 H),
2-oxo-1,2- 7.51-7.39 (m, 1 H), 7.17-6.98 (m, 2 H), 6.75 (d, J = 8.5
Hz, dihydropyridin-3- 1 H), 6.61-6.49 (m, 1 H), 6.15 (s, 1 H),
yl)methyl)-2- 4.49-4.38 (m, 1 H), 4.33 (d, J = 3.8 Hz, 2 H),
methyl-1-(1-(1- 4.24-4.03 (m, 2 H), 3.85 (s, 3 H), (pyridin-2-
2.90-2.70 (m, 2 H), 2.58 (s, 3 H), 2.20 (s, 3 H), yl)piperidin-4-
2.06-1.91 (m, 1 H), 1.63-1.47 (m, 4 H), 1.40-1.27 (m, 1
yl)ethyl)-1H- H), 1.07-0.94 (m, 1 H), 0.82-0.72 (m, 1 H) indole-3-
carboxamide
Example 5
Synthesis of (R or
S)-1-(1-(1-(2-hydroxyethyl)piperidin-4-yl)ethyl)-N-((4-methoxy-6-methyl-2-
-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methyl-1H-indole-3-carboxamide
(Compound 347)
##STR00038##
[0100] To a sealed tube charged with a magnetic stir bar was added
(R or
S)--N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methyl-
-1-(1-(piperidin-4-yl)ethyl)-1H-indole-3-carboxamide (0.1 g, 0.229
mmol) was added DCM (3 mL) and the reaction cooled to 0.degree. C.
To the cooled reaction mixture was added oxirane which was
condensed into the reaction vial (.about.1 mL). The reaction was
allowed to stir to rt over 4 h and was then conc. in vacuo to
afford the crude material which was purified via silica gel
chromatography (12 g) using ethyl acetate/MeOH (4:1) as eluent to
afford the title compound as a white solid (50 mg). LCMS 481
(M+1).sup.+; .sup.1H NMR (DMSO-d6, 400 MHz) .delta.) .delta. 11.58
(s, 1H), 7.77-7.65 (m, 2H), 7.59 (d, J=7.8 Hz, 1H), 7.11-6.99 (m,
2H), 6.14 (s, 1H), 4.54-4.44 (m, 1H), 4.31 (d, J=5.1 Hz, 3H), 4.13
(dd, J=7.1, 10.3 Hz, 1H), 3.83 (s, 3H), 3.42 (q, J=6.0 Hz, 2H),
2.93 (br. s., 1H), 2.71-2.56 (m, 4H), 2.31 (br. s., 2H), 2.19 (s,
3H), 2.03-1.83 (m, 2H), 1.64 (br. s., 1H), 1.53 (d, J=6.9 Hz, 3H),
1.32 (d, J=11.1 Hz, 1H), 1.02 (d, J=10.3 Hz, 1H), 0.65 (d, J=11.8
Hz, 1H).
[0101] The compounds shown in the following table were prepared
according to the general procedure outlined in this Example using
the appropriate starting materials. The structures of the compounds
are shown in FIG. 1.
TABLE-US-00014 Compound Number Name .sup.1H NMR m/z 352 (R or
S)-1-(1-(1-(2- NMR (400 MHz, DMSO-d6) .delta. 11.58 (br. s., 1 509
hydroxy-2- H), 7.76-7.65 (m, 2 H), 7.58 (d, J = 7.8 Hz, 1
methylpropyl)piperidin- H), 7.10-6.99 (m, 2 H), 6.14 (s, 1 H), 4.31
(d, 4-yl)ethyl)-N-((4- J = 4.9 Hz, 2 H), 4.14 (br. s., 1 H), 3.94
(s, 1 methoxy-6-methyl-2- H), 3.83 (s, 3 H), 3.56 (s, 2 H), 3.01
(d, J = 11.4 Hz, oxo-1,2- 1 H), 2.73-2.64 (m, 1 H), 2.59 (s, 3
dihydropyridin-3- H), 2.19 (s, 3 H), 2.16-2.03 (m, 2 H), 1.52 (d,
yl)methyl)-2-methyl- J = 6.9 Hz, 4 H), 1.34 (br. s., 2 H), 1.02 (d,
J = 4.5 Hz, 1H-indole-3- 7 H), 0.66-0.58 (m, 1 H) carboxamide 369
(R or S)-N-((4,6- (400 MHz, DMSO-d6) .delta. 11.59 (br. s., 1 H),
493 dimethyl-2-oxo-1,2- 7.77-7.69 (m, 1 H), 7.60 (br. s., 2 H),
dihydropyridin-3- 7.06 (br. s., 2 H), 5.89 (s, 1 H), 4.31 (t, J =
5.7 Hz, 2 yl)methyl)-1-(1-(1-(2- H), 4.20-4.09 (m, 1 H), 4.00-3.92
(m, 1 H), hydroxy-2- 3.58-3.55 (m, 2 H), 3.19-3.10 (m, 1 H),
methylpropyl)piperidin- 3.07-2.95 (m, 1 H), 2.74-2.63 (m, 1 H),
2.59 (br. 4-yl)ethyl)-2-methyl- s., 3 H), 2.27 (s, 3 H), 2.12 (s, 3
H), 1H-indole-3- 1.89-1.72 (m, 1 H), 1.54 (br. s., 4 H), 1.30-1.14
(m, 2 carboxamide H), 1.03 (br. s., 6 H), 0.84-0.57 (m, 2 H)
Example 6
Synthesis of (R or
S)--N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methyl-
-6-phenyl-1-(1-(tetrahydro-2H-pyran-4-yl)ethyl)-1H-indole-3-carboxamide
(Compound 374)
##STR00039##
[0102] A 25 mL reaction tube was charged with a magnetic stir bar,
phenyl boronic acid (72.6 mg, 0.596 mmol), K.sub.3PO.sub.4 (103 mg,
0.447 mmol), X-Phos pre-catalyst
(Chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2-
-aminoethyl)phenyl)]palladium(II)) (4.92 mg, 5.96 .mu.mol), and the
vial was sealed. The vial was evacuated/backfilled with nitrogen
(3.times.) before the addition of methyl
6-chloro-2-methyl-1-(1-(tetrahydro-2H-pyran-4-yl)ethyl)-1H-indole-3-carbo-
xylate (Compound 314) (100 mg, 0.298 mmol) as a solution in
1,4-dioxane (1 mL). The vial was then heated to 100.degree. C.
overnight with stirring. The vial was then allowed to cool to room
temperature and the reaction concentrated in vacuo. The crude
residue was purified via SiO.sub.2 chromatography (10 g) using an
eluent of ethyl acetate/hexanes (4:1) the title compound as a white
solid (106 mg, 0.281 mmol, 94% yield).). LCMS 514 (M+1).sup.+;
.sup.1H NMR .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 11.59 (s,
1H), 7.98-7.84 (m, 2H), 7.75-7.67 (m, 3H), 7.47 (t, J=7.8 Hz, 2H),
7.39 (d, J=8.5 Hz, 1H), 7.35-7.27 (m, 1H), 6.15 (s, 1H), 4.35 (d,
J=4.9 Hz, 2H), 4.25-4.12 (m, 1H), 3.93 (d, J=8.5 Hz, 1H), 3.86-3.77
(m, 3H), 3.67 (d, J=8.5 Hz, 1H), 3.39-3.32 (m, 1H), 3.10-3.00 (m,
1H), 2.62 (s, 3H), 2.21 (s, 3H), 1.85 (d, J=10.0 Hz, 1H), 1.63-1.49
(m, 4H), 1.45-1.33 (m, 1H), 1.20-0.99 (m, 1H), 0.66 (d, J=12.0 Hz,
1H).
Example 7
Synthesis of (R or
S)-2-(4-(1-(3-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methylca-
rbamoyl)-2-methyl-1H-indol-1-yl)ethyl)piperidin-1-yl) acetic acid
(Compound 364)
##STR00040##
[0103] To a round bottomed flask was charged with a magnetic stir
bar was added (R or
S)-ethyl-2-(4-(1-(3-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)me-
thylcarbamoyl)-2-methyl-1H-indol-1-yl)ethyl)piperidin-1-yl)acetate
(Compound 363) (69 mg, 0.132 mmol), THF (1.5 mL), MeOH (1.5 mL),
and water (0.75 mL). To this solution was added lithium hydroxide
monohydrate (5.54 mg, 0.132 mmol) and the reaction stirred at room
temperature for 1 h. The organics were removed under reduced
pressure and the resulting aqueous solution purified via reverse
phase-HPLC (water/MeCN) 0.fwdarw.95% to afford the title compound
(66 mg, 0.108 mmol, 82% yield). LCMS 514 (M+1).sup.+ 1H NMR (400
MHz, DMSO-d.sub.6) .delta.=11.67 (s, 1H), 9.65 (s, 1H), 7.84-7.68
(m, 2H), 7.63 (d, J=7.4 Hz, 1H), 7.14-7.03 (m, 2H), 6.18 (s, 1H),
4.33 (d, J=3.6 Hz, 2H), 4.27-4.15 (m, 1H), 4.04 (br. s., 2H), 3.85
(s, 3H), 3.57 (s, 1H), 3.35-3.23 (m, 1H), 3.14-2.99 (m, 1H),
2.86-2.74 (m, 1H), 2.62 (s, 3H), 2.21 (s, 3H), 2.18-2.08 (m, 1H),
1.75 (s, 1H), 1.60-1.49 (m, 4H), 1.46-1.33 (m, 1H), 0.92-0.81 (m,
1H).
Example 8
Synthesis of (R or S)-methyl
2-methyl-6-(pyridin-3-yl)-1-(1-(tetrahydro-2H-pyran-4-yl)ethyl)-1H-indole-
-3-carboxylate
[0104] This intermediate was used as an alternate starting material
in Step 7 set forth in Example 1 for the synthesis of other
compounds of the invention.
(R or S)-methyl
2-methyl-1-(1-(tetrahydro-2H-pyran-4-yl)ethyl)-6-(4,4,5,5-tetramethyl-1,3-
,2-dioxaborolan-2-yl)-1H-indole-3-carboxylate
##STR00041##
[0105] To a round bottomed flask was added Pd(OAc).sub.2 (10.03 mg,
0.045 mmol), potassium acetate (219 mg, 2.233 mmol),
4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane) (567
mg, 2.233 mmol), and
2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (XPhos) (85
mg, 0.179 mmol), and the vial was sealed. To this vessel was added
(R or S)-methyl
6-chloro-2-methyl-1-(1-(tetrahydro-2H-pyran-4-yl)ethyl)-1H-indole-3-carbo-
xylate (Step 6) (500 mg, 1.489 mmol) dissolved in dioxane (3.4 mL)
and the reaction evacuated/back-filled with N.sub.2 (3.times.)
before heating to 100.degree. C. overnight. The reaction was then
allowed to cool to rt and was diluted with EtOAc. The reaction was
filtered through diatomaceous earth and the filtrate concentrated
to afford the title compound which was used in subsequent reactions
without further purification. LCMS 428 (M+1).sup.+.
(R or S)-methyl
2-methyl-6-(pyridin-3-yl)-1-(1-(tetrahydro-2H-pyran-4-yl)ethyl)-1H-indole-
-3-carboxylate
##STR00042##
[0106] To a re-sealable vial was added K.sub.2CO.sub.3 (206 mg,
1.488 mmol), PdCl.sub.2(dppf)-CH.sub.2Cl.sub.2 adduct (60.8 mg,
0.074 mmol), and the vial was sealed. This vial was
evacuated/backfilled with N.sub.2 (3.times.) before addition of (R
or S)-methyl
2-methyl-1-(1-(tetrahydro-2H-pyran-4-yl)ethyl)-6-(4,4,5,5-tetramethyl-1,3-
,2-dioxaborolan-2-yl)-1H-indole-3-carboxylate (318 mg, 0.744 mmol)
dissolved in 1,4-dioxane (4 mL), 3-bromopyridine (71.7 .mu.l, 0.744
mmol), and water (400 .mu.L). The reaction was evacuated/backfilled
with N.sub.2 (3.times.) before heating to 100.degree. C. The
solution was cooled to room temperature and diluted with EtOAc. The
solution was filtered and concentrated in vacuo. The crude residue
was purified via silica gel chromatography (10 g, EtOAc/hex (1:1))
to afford the title compound (101 mg, 0.267 mmol, 35.9% yield).
LCMS 379 (M+1).sup.+.
Example 9
Other Alkyl Carboxylate Intermediates
[0107] The following alkyl carboxylate intermediates were
synthesized in an analogous manner to that set forth in Step 2 of
Example 1, using an appropriate starting material and reactant.
TABLE-US-00015 Name Structure m/z (.+-.)-ethyl 5-fluoro-1-
(1-methoxypropan-2- yl)-2-methyl-1H- indole-3-carboxylate
##STR00043## 294 (.+-.)-ethyl 6-fluoro-1- (1-methoxypropan-2-
yl)-2-methyl-1H- indole-3-carboxylate ##STR00044## 294 (.+-.)-ethyl
1-(1- methoxypropan-2- yl)-2-methyl-1H- indole-3-carboxylate
##STR00045## 276 (.+-.)-tert-butyl 1-(1- methoxypropan-2-
yl)-2-methyl-1H- pyrrolo[2,3-b] pyridine-3- carboxylate
##STR00046## 305 (.+-.)-tert-butyl 1-(1- ethoxypropan-2-
yl)-2-methyl-1H- pyrrolo[2,3- b]pyridine-3- carboxylate
##STR00047## 319 tert-butyl 1-(3- methoxybutan-2- yl)-2-methyl-1H-
pyrrolo[2,3- b]pyridine-3- carboxylate ##STR00048## 319 ethyl 1-(3-
methoxybutan-2- yl)-2-methyl-6- (methylsulfonyl)- 1H-indole-3-
carboxylate ##STR00049## 368 (.+-.)-ethyl 1-(3- methoxypentan-
2-yl)-2-methyl- 1H-indole-3- carboxylate ##STR00050## 304
Example 10
Other Compounds of the Invention Produced from Carboxylic Acid
Intermediates
[0108] The following compounds were synthesized in an analogous
manner to that set forth in Step 4 of Example 1, using an
appropriate starting material. Structures of these compounds are
set forth in FIG. 1.
TABLE-US-00016 Compound Name .sup.1H NMR m/z 304 (.+-.)-1-(1-(4,4-
(CDCl.sub.3, 400 MHz) .delta. 12.63-12.64 (d, J = 3.2 Hz, 427
difluorocyclohexyl)ethyl)- 1H), 7.84 (s, 1H), 7.49 (s, 1H),
N-((4-methoxy-6-methyl- 7.42-7.40 (d, J = 9.2 Hz, 1H), 7.06-7.00
(m, 2H), 2-oxo-1,2-dihydropyridin- 5.90-5.89 (d, J = 3.6 Hz 1H),
4.66-4.62 (t, J = 14 Hz, 3-yl)methyl)-2-methyl- 2H), 4.11-4.08 (m,
1H), 3.88-3.87 (d, J = 3.6 Hz, 1H-indole-3-carboxamide 3H),
2.99-2.76 (m, 3H), 2.36 (s, 1H), 2.25 (s, 3H), 2.17-2.16 (d, J =
3.2 Hz, 2H), 2.08-2.05 (m, 2H), 1.84-1.70 (m, 2H), 1.61 (s, 1H),
1.51-1.47 (m, 2H) 230 (.+-.)-5-fluoro-N-((4- (400 MHz, CD.sub.3OD)
.delta. 7.59-7.55 (m, 1H), 416 methoxy-6-methyl-2-oxo- 7.42-7.39
(m, 1H), 6.95-6.90 (m, 2H), 1,2-dihydropyridin-3- 4.57 (s, 2H),
4.12 (s, 3H), 3.99-3.94 (m, 1H), yl)methyl)-1-(1- 3.72-3.65 (m,
1H), 3.19 (s, 3H), 2.64 (s, 3H), methoxypropan-2-yl)-2- 2.54 (s,
3H), 1.59-1.57 (d, 3H) methyl-1H-indole-3- carboxamide 231
(.+-.)-6-fluoro-N-((4- (400 MHz, CD.sub.3OD) .delta. 7.70-7.66 (m,
1H), 416 methoxy-6-methyl-2-oxo- 7.36-7.33 (m, 1H), 6.94-6.89 (m,
2H), 1,2-dihydropyridin-3- 4.56 (s, 2H), 4.11 (s, 3H), 3.97-3.92
(m, 1H), yl)methyl)-1-(1- 3.71-3.67 (m, 1H), 3.20 (s, 3H), 2.62 (s,
3H), methoxypropan-2-yl)-2- 2.53 (s, 3H), 1.58-1.56 (d, 3H)
methyl-1H-indole-3- carboxamide 218 (.+-.)-N-((4-methoxy-6- (400
MHz, CD.sub.3OD) .delta. 7.69 (d, J = 7.2 Hz, 398 methyl-2-oxo-1,2-
1H), 7.53 (d, J = 7.6 Hz, 1H), 7.12 (m, dihydropyridin-3- 2H), 6.26
(s, 1H), 4.80 (m, 1H), 4.52 (s, 2H), yl)methyl)-1-(1- 3.99 (m, 4H),
3.75 (m, 1H), 3.20 (s, 3H), methoxypropan-2-yl)-2- 2.62 (s, 3H),
2.31 (s, 3H), 1.59 (d, J = 7.2 Hz, 3H) methyl-1H-indole-3-
carboxamide 183 (.+-.)-N-((4,6-dimethyl-2- (400 MHz, CD.sub.3OD)
.delta. 7.74 (m, 1H), 382 oxo-1,2-dihydropyridin-3- 7.57 (d, J =
7.6 Hz, 1H), 7.15 (m, 2H), yl)methyl)-1-(1- 6.14 (s, 1H), 4.86 (m,
1H), 4.55 (s, 2H), 4.02 (m, methoxypropan-2-yl)-2- 1H), 3.77 (m,
1H), 3.22 (s, 3H), 2.65 (s, 3H), methyl-1H-indole-3- 2.43 (s, 3H),
2.26 (s, 3H), 1.62 (d, J = 7.2 Hz, carboxamide 3H) 204
(.+-.)-N-((4-methoxy-6- (400 MHz, CDCl.sub.3) .delta. 13.23 (s,
1H), 399 methyl-2-oxo-1,2- 8.16-8.17 (m, 1H), 8.11-8.13 (m, 1H),
dihydropyridin-3- 7.57-7.60 (t, J = 5.2 Hz, 1H), 6.93-6.96 (m, 1H),
yl)methyl)-1-(1- 5.92 (s, 1H), 4.82-4.83 (d, J = 2.4 Hz, 1H),
methoxypropan-2-yl)-2- 4.65-4.66 (d, J = 6.4 Hz, 2H), 3.89 (s, 3H),
methyl-1H-pyrrolo[2,3- 3.81-3.85 (m, 1H), 3.22 (s, 3H), 2.79 (s,
3H), b]pyridine-3-carboxamide 2.17 (s, 3H), 1.64-1.66 (d, J = 8.0
Hz, 3H) 211 (.+-.)-1-(1- 379 cyclopropylethyl)-N-((4,6-
dimethyl-2-oxo-1,2- dihydropyridin-3- yl)methyl)-2-methyl-1H-
pyrrolo[2,3-b]pyridine-3- carboxamide 212
(.+-.)-1-(1-ethoxypropan-2- (400 MHz, CDCl.sub.3) .delta.
8.173-8.189 (m, 1H), 413 yl)-N-((4-methoxy-6- 8.13-8.153 (m, 1H),
7.563 (s, 1H), methyl-2-oxo-1,2- 6.977-7.008 (m, 1H), 5.938 (s,
1H), dihydropyridin-3- 4.652-4.667 (d, 2H), 4.177 (s, 1H),
3.309-3.454 (m, 2H), yl)methyl)-2-methyl-1H- 3.94-3.98 (m, 1H),
2.806 (s, 3H), 2.212 (s, pyrrolo[2,3-b]pyridine-3- 3H), 1.665-1.682
(d, 3H), 1.044 (t, 3H) carboxamide 235 (.+-.)-N-((4-ethoxy-6- (400
MHz, CDCl.sub.3) .delta. 413 methyl-2-oxo-1,2- 12.5 (s, 1H),
8.11-8.18 (m, 2H), 7.60 (s, dihydropyridin-3- 1H), 6.95-6.98 (m,
1H), 5.90 (s, 1H), yl)methyl)-1-(1- 5.96 (s, 1H), 4.83 (s, 1H),
4.10-4.21 (m, 3H), methoxypropan-2-yl)-2- 3.82-3.83 (m, 1H), 3.23
(s, 3H), 3.79 (s, methyl-1H-pyrrolo[2,3- 3H), 2.15 (s, 3H),
1.65-1.66 (d, J = 6.8 Hz, b]pyridine-3-carboxamide 6H), 1.44-1.47
(t, J = 7.2 Hz, 3H). 241 N-((4,6-dimethyl-2-oxo- (400 MHz,
CD.sub.3OD): .delta. 8.67-8.65 (d, 1H), 397 1,2-dihydropyridin-3-
8.45-8.44 (d, 1H), 7.59-7.55 (m, 1H), yl)methyl)-1-(3- 6.70 (s,
1H), 4.79 (s, 1H), 4.61 (s, 2H), 4.07 (s, methoxybutan-2-yl)-2-
1H), 3.32 (s, 3H), 2.75 (s, 3H), 2.56 (s, 3H),
methyl-1H-pyrrolo[2,3- 2.42 (s, 3H), 1.68-1.66 (d, 3H), 1.16-1.15
(d, b]pyridine-3-carboxamide 3H). 280 (.+-.)-N-((6-ethyl-4- .sup.1H
NMR (400 MHz, CD.sub.3OD) .delta. 413 methoxy-2-oxo-1,2- 8.25-8.29
(m, 2H). .delta. 7.28-7.31 (m, 1H). 6.89 (s, 1H), dihydropyridin-3-
4.93-4.95 (br, 1H), 4.58 (s, 2H), yl)methyl)-1-(1- 4.2-4.25 (m,
1H), 4.13 (s, 3H), 3.77-3.81 (m, 1H), methoxypropan-2-yl)-2- 3.24
(s, 3H), 2.79-2.84 (q, 1H), 2.72 (s, 3H), methyl-1H-pyrrolo[2,3-
1.66-1.68 (d, J = 7.2 Hz, 3H) 1.32-1.36 (t,
b]pyridine-3-carboxamide 3H) 288 (R or S)-N-((4-Methoxy- (400 MHz,
d6-DMSO) .delta. 11.57-11.65 (m, 439 6-methyl-2-oxo-1,2- 1H),
8.18-8.23 (m, 1H), 8.07-8.12 (m, dihydropyridin-3- 1H), 7.83-7.91
(m, 1H), 7.07-7.15 (m, yl)methyl)-2-methyl-1-(1- 1H), 6.15 (s, 1H),
4.31 (d, J = 4.46 Hz, 1H), (tetrahydro-2H-pyran-4- 4.04-4.20 (m,
1H), 3.88-3.97 (m, 1H), yl)ethyl)-1H-pyrrolo[2,3- 3.84 (s, 3H),
3.59-3.70 (m, 1H), b]pyridine-3-carboxamide 2.97-3.10 (m, 1H),
2.79-2.93 (m, 1H), 2.67 (br. S., 3H), 2.20 (s, 3H), 1.78-1.88 (m,
1H), 1.53-1.68 (m, 3H), 1.28-1.41 (m, 2H), 0.97-1.13 (m, 2H),
0.56-0.68 (m, 1H) 306 (R or S)-N-((4,6-dimethyl- (400 MHz,
DMSO-d.sub.6) .delta. = 11.73-11.56 (m, 423
2-oxo-1,2-dihydropyridin- 1 H), 8.19 (d, J = 3.1 Hz, 1 H), 8.06
(dd, J = 1.4, 3-yl)methyl)-2-methyl-1- 7.9 Hz, 1 H), 7.82 (br. S.,
1 H), (1-(tetrahydro-2H-pyran- 7.10 (dd, J = 4.7, 7.8 Hz, 1 H),
5.91 (s, 1 H), 4-yl)ethyl)-1H-indole-3- 4.30 (br. S., 2 H),
4.19-4.02 (m, 1 H), 3.90 (d, J = 8.5 Hz, carboxamide 1 H), 3.63 (d,
J = 7.8 Hz, 1 H), 3.29 (s, 1 H), 3.06 (s, 1 H), 2.92-2.74 (m, 1 H),
2.64 (br. S., 3 H), 2.25 (s, 3 H), 2.11 (s, 3 H), 1.80 (br. S., 1
H), 1.59 (br. S., 3 H), 1.41-1.24 (m, 1 H), 1.09 (s, 2 H),
0.67-0.52 (m, 1 H) 277 (.+-.)-1-(3-methoxy-3- (400 MHz,
DMSO-d.sub.6) .delta. = 12.01-11.82 (m, 426
methylbutan-2-yl)-N-((4- 1 H), 7.91-7.82 (m, 2 H), 7.71-7.64 (m, 1
methoxy-6-methyl-2-oxo- H), 7.06-6.96 (m, 2 H), 6.25 (s, 1 H),
1,2-dihydropyridin-3- 4.43 (q, J = 7.1 Hz, 1 H), 4.33 (br. S., 2
H), yl)methyl)-2-methyl-1H- 3.86 (s, 3 H), 3.14-3.09 (m, 3 H), 2.61
(s, 3 H), indole-3-carboxamide 2.23 (s, 3 H), 1.58-1.52 (m, 3 H),
1.27 (s, 3 H), 0.88 (s, 3 H) 275 (.+-.)-N-((4,6-dimethyl-2- 410
oxo-1,2-dihydropyridin-3- yl)methyl)-1-(3- methoxypentan-2-yl)-2-
methyl-1H-indole-3- carboxamide 294 (.+-.)-N-((4-methoxy-6-
(CDCl.sub.3, 400 MHz) .delta. 7.85 (t, J = 6.4 Hz, 412
methyl-2-oxo-1,2- 1H), 7.45 (s, 2H), 7.08-7.03 (m, 2H),
dihydropyridin-3- 5.93 (s, 1H), 4.71-4.61 (m, 2H), 4.36 (s, 1H),
yl)methyl)-1-(3- 3.90 (s, 4H), 2.95 (s, 3H), 2.75 (s, 3H),
methoxybutan-2-yl)-2- 2.17 (s, 3H), 1.57 (d, J = 7.2 Hz, 3H), 1.23
(d, J = 6.0 Hz, methyl-1H-indole-3- 3H) carboxamide 290
(.+-.)-1-(3-ethoxybutan-2- (400 MHz, DMSO-d.sub.6) .delta. = 11.60
(br. s., 1 426 yl)-N-((4-methoxy-6- H), 7.72 (d, J = 7.6 Hz, 1 H),
7.67 (d, J = 5.1 Hz, methyl-2-oxo-1,2- 2 H), 7.09-6.98 (m, 2 H),
6.14 (s, 1 dihydropyridin-3- H), 4.41-4.35 (m, 1 H), 4.32 (d, J =
4.9 Hz, yl)methyl)-2-methyl-1H- 2 H), 4.03-3.93 (m, 1 H), 3.83 (s,
3 H), indole-3-carboxamide 3.25 (d, J = 9.4 Hz, 1 H), 2.82-2.72 (m,
1 H), 2.62 (br. s., 3 H), 2.19 (s, 3 H), 1.52 (d, J = 7.1 Hz, 3 H),
1.15 (d, J = 6.0 Hz, 3 H), 0.68 (t, J = 6.9 Hz, 3 H) 293
N-((4-methoxy-6-methyl- (400 MHz, CDCl.sub.3): .delta. 8.19-8.13
(m, 2H), 427 2-oxo-1,2-dihydropyridin- 7.57-7.55 (t, 1H), 6.99-6.96
(m, 1H), 3-yl) methyl)-1-(3- 5.94 (s, 1H), 4.67-4.65 (m, 2H), 4.40
(m, 1H), methoxypentan-2-yl)-2- 4.16 (m, 1H), 3.16 (s, 3H), 2.80
(s, 3H), methyl-1H-pyrrolo [2,3- 2.77 (s, 3H), 2.20 (s, 3H),
1.87-1.81 (m, b] pyridine-3-carboxamide 1H), 1.67-1.65 (m, 3H),
1.53-1.45 (m, 3H), 1.02-0.99 (m, 3H) 299 N-((4-methoxy-6-methyl-
(400 MHz, CDCl.sub.3) .delta. 7.87-7.86 (d, 1H), 425
2-oxo-1,2-dihydropyridin- 7.52-7.45 (m, 2H), 7.10-7.02 (m, 2H),
3-yl)methyl)-1-(3- 4.72-4.64 (dd, 2H), 4.45-4.42 (s 1H), 3.9 (s,
3H), methoxypentan-2-yl)-2- 3.73 (s, 1H), 2.8-2.7 (d, 6H), 2.17 (s,
3H), methyl-1H-indole-3- 1.80-1.75 (m, 1H), 1.58 (s, 3H),
carboxamide 1.25 (m, 1H), 1.03-0.99 (t, 3H)
Example 11
Synthesis of Methyl
1-(1-(1,4-dioxan-2-yl)ethyl)-2-methyl-1H-indole-3-carboxylate
[0109] The title compound was used as an alternate alkyl
carboxylate starting material in Step 3 of Example 1.
Step 1: 1-(1,4-dioxan-2-yl)ethanone
##STR00051##
[0110] To a solution of benzoic peroxide (20 g, 141 mmol) in 200 mL
1,4-dioxane at room temperature under nitrogen atmosphere was added
biacetyl (24.3 g, 282 mmol). After the addition, the mixture was
heated to reflux and stirred for 24 hours. The reaction mixture was
cooled to 0.degree. C. The pH was adjusted to around 9 by
progressively adding 2N sodium hydroxide below 0.degree. C.,
extracted with 2-methoxy-2-methylpropane (10 mL.times.3), and
concentrated to give 1-(1,4-dioxan-2-yl)ethanone (13 g, 36%) as a
yellow oil which was used directly in the next step without
purification.
Step 2: 1-(1,4-dioxan-2-yl)ethanamine
##STR00052##
[0111] To a solution of 1-(1,4-dioxan-2-yl)ethanone (12 g, 92.2
mmol) in 1,2-dichloroethane (100 mL) was added
(4-methoxyphenyl)methanamine (25 g, 184.4 mmol) at room
temperature. The mixture was allowed to stir for 3 hours, and then
sodium triacetoxyborohydride (39 g, 184.4 mmol) was added. The
resulting mixture was allowed to stir for 48 hours at room
temperature. The reaction mixture was quenched by adding water,
extracted with dichloromethane (100 mL.times.3). The combined
organic phase was dried by anhydrous sodium sulphate, and then
filtered. The filtrate was concentrated and purified by column
chromatograph on silica gel (elute:dichloromethane/methanol
100:1.fwdarw.50:1.fwdarw.20:1) to give
1-(1,4-dioxan-2-yl)-N-(4-methoxybenzyl)ethanamine (16.4 g, 71%) as
a yellow solid. LCMS (M+H.sup.+) m/z: calcd. 251.15. found 251.9.
To a solution of 1-(1,4-dioxan-2-yl)-N-(4-methoxybenzyl)ethanamine
(5 g, 19.9 mmol) in anhydrous methanol (100 mL) was added palladium
10% on carbon (240 mg, 2 mmol), then purged with hydrogen (30 psi),
the mixture was allowed to stir overnight at room temperature. The
reaction mixture was filtered, and the filtrate was concentrated to
afford the title compound (2.5 g, 96%) as a brown solid.
[0112] The amine intermediates shown in the following table were
prepared according to the general procedure outlined above using
the appropriate starting materials and modifications.
TABLE-US-00017 Name Structure m/z tert-butyl
3-(1-aminoethyl)piperidine- 1-carboxylate ##STR00053## 228
(.+-.)-1-(4,4-difluorocyclohexyl) ethanamine ##STR00054## 164
(.+-.)-1-(1-(methylsulfonyl)azetidin- 3-yl)ethanamine ##STR00055##
179 (.+-.)-tert-butyl 4-(4-(1- aminoethyl)47yridine-
2-yl)piperazine-1-carboxylate ##STR00056## 307
Step 3: (E)-methyl
3-((1-(1,4-dioxan-2-yl)ethyl)imino)-2-(2-bromophenyl)butanoate
##STR00057##
[0113] To a solution of 1-(1,4-dioxan-2-yl)ethanamine (2.5 g, 19
mmol) in methanol (100 mL) was added methyl
2-(2-bromophenyl)-3-oxobutanoate (5.4 g, 20 mmol) and acetic acid
(1.8 g, 30 mmol). The resulting reaction system was warm to reflux
and allowed to stir overnight. The reaction mixture was
concentrated and purified by column chromatographed on silica gel
(eluted:dichloromethane/methanol 50:1.fwdarw.20:1.fwdarw.5:1) the
title compound (1 g, 14%) as a brown solid. LCMS (M+H.sup.+) m/z:
calcd. 383.07. found 384.9.
[0114] The imino-bromo intermediates shown in the following table
were prepared according to the general procedure outlined above
using the appropriate starting materials (e.g., one of the amines
set forth in the table in Step 2 of this example) and
modifications.
TABLE-US-00018 Name Structure m/z (E)-tert-butyl 3- (1-((3-(2-
bromophenyl)- 4-methoxy-4- oxobutan-2- ylidene)amino)
ethyl)piperidine- 1-carboxylate ##STR00058## 482 (.+-.)-(E)-methyl
2- (2-bromophenyl)- 3-((1-(4,4- difluoro- cyclohexyl) ethyl)imino)
butanoate ##STR00059## 417 (E)-tert-butyl 4- ((3-(2- bromophenyl)-
4-methoxy-4- oxobutan-2- ylidene)amino) piperidine-1- carboxylate
##STR00060## 454 (Z)-methyl 2-(2- bromophenyl)-3- (quinolin-5-
ylamino)but-2- enoate ##STR00061## 398 (E)-methyl 2-(2-
bromophenyl)-3- (cyclo- pentylimino) butanoate ##STR00062## 339
(E)-methyl 2-(2- bromophenyl)-3- ((6- methylquinolin- 5-yl)imino)
butanoate ##STR00063## 412 (.+-.)-(E)-methyl 2- (2-bromophenyl)-
3-((1-(1- (methylsulfonyl) azetidin-3- yl)ethyl)imino) butanoate
##STR00064## 432 (.+-.)-(E)-tert-butyl 4-(4-((3-(2- bromophenyl)-4-
methoxy-4- oxobutan-2- ylidene)amino) pyridine-2- yl)piperazine-1-
carboxylate ##STR00065## 559 (E)-methyl 2-(2- bromophenyl)-3-
((2,5- dimethylphenyl) amino)but-2- enoate ##STR00066## 375
(E)-methyl 2-(2- bromophenyl)-3- ((2,3- dimethylphenyl)
amino)but-2- enoate ##STR00067## 375 (E)-methyl 2-(2-
bromophenyl)-3- (quinolin-6- ylimino)butanoate ##STR00068## 398
Step 4: Methyl
1-(1-(1,4-dioxan-2-yl)ethyl)-2-methyl-1H-indole-3-carboxylate
##STR00069##
[0115] To a solution of (E)-methyl
3-((1-(1,4-dioxan-2-yl)ethyl)imino)-2-(2-bromophenyl)butanoate (400
mg, 1.1 mmol) in dioxane (3 mL) was added
Chloro[2-(dicyclohexylphosphino)-3,6-dimethoxy-2',4',6'-triisopropylbiphe-
nyl][2-(2-aminoethyl)phenyl]Pd(II) (160 mg, 0.2 mmol),
2-Dicyclohexyphosphino-2',6'-diisopropoxybiphenyl (93 mg, 0.2 mmol)
and sodium tert-butoxide (192 mg, 2 mmol). The resulting reaction
mixture was heated to 120.degree. C. with stirring for 30 mins in a
microwave. The reaction mixture was quenched by adding water and
was extracted with ethyl acetate (25 mL.times.3). The combined
organic phase was dried by anhydrous sodium sulphate, and then
filtered. The filtrate was concentrated and purified by column
chromatograph on silica gel (eluted:petrol ether/acetic ester
10:1.fwdarw.5:1.fwdarw.2:1) to afford the title compound (282 mg,
89%) as yellow solid. LCMS (M+H.sup.+) m/z: calcd. 303.15. found
303.9.
[0116] The compound shown in the following table was prepared
according to the general procedure outlined above using the
appropriate starting materials (e.g., one of the imino-bromo
intermediates shown in the table in Step 3 of this example) and
modifications.
TABLE-US-00019 Name Structure m/z (.+-.)-methyl 1-(1-(4,4-
difluorocyclohexyl) ethyl)-2-methyl- 1H-indole-3- carboxylate
##STR00070## 336
[0117] These alkyl carboxylates were also used as starting material
in Step 3 of Example 1 in the synthesis of certain compounds of the
invention.
Example 12
Chiral Separation of Compound 219 to afford Compounds 223 and
224
[0118]
N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-1-(1-m-
ethoxypropan-2-yl)-2-methyl-1H-indole-3-carboxamide (200 mg)
(Compound 219) was subjected to chiral chromatography via
supercritical fluid chromatography (SFC)
(A:C.sub.2H.sub.5OH,B:NH.sub.3.H.sub.2O. A:B=55:45 AD column) to
afford the separate enantiomers 223 (peak 1) and 224 (Peak 2) (60
mg each) LCMS 398 (M+1).sup.+ 1H NMR (400 MHz, CD.sub.3OD) .delta.
7.69 (d, J=7.2 Hz, 1H), 7.53 (d, J=7.6 Hz, 1H), 7.12 (m, 2H), 6.26
(s, 1H), 4.80 (m, 1H), 4.52 (s, 2H), 3.99 (m, 4H), 3.75 (m, 1H),
3.20 (s, 3H), 2.62 (s, 3H), 2.31 (s, 3H), 1.59 (d, J=7.2 Hz, 3H).
The optical rotation of each enantiomer was not determined.
[0119] The compounds shown in the following table were prepared
according to the general chiral chromatography procedure outlined
above. The optical rotation of the separated enantiomers was not
determined, but the elution peak ("Peak 1" or "Peak 2") is
indicated. Structures of each compound are shown in FIG. 1.
TABLE-US-00020 Compound Name .sup.1H NMR m/z 217 (R or
S)-N-((4,6-dimethyl-2- (400 MHz, CD.sub.3OD) .delta. 7.74 (m, 1H),
382 oxo-1,2-dihydropyridin-3- 7.57 (d, J = 7.6 Hz, 1H), 7.15 (m,
2H), yl)methyl)-1-(1- 6.14 (s, 1H), 4.86 (m, 1H), 4.55 (s, 2H),
methoxypropan-2-yl)-2- 4.02 (m, 1H), 3.77 (m, 1H), 3.22 (s,
methyl-1H-indole-3- 3H), 2.65 (s, 3H), 2.43 (s, 3H), 2.26 (s,
carboxamide-PEAK 1 3H), 1.62 (d, J = 7.2 Hz, 3H) 218 (R or
S)-N-((4,6-dimethyl-2- (400 MHz, CD.sub.3OD) .delta. 7.74 (m, 1H),
382 oxo-1,2-dihydropyridin-3- 7.57 (d, J = 7.6 Hz, 1H), 7.15 (m,
2H), yl)methyl)-1-(1- 6.14 (s, 1H), 4.86 (m, 1H), 4.55 (s, 2H),
methoxypropan-2-yl)-2- 4.02 (m, 1H), 3.77 (m, 1H), 3.22 (s, 3H),
methyl-1H-indole-3- 2.65 (s, 3H), 2.43 (s, 3H), 2.26 (s, 3H),
carboxamide-PEAK 2 1.62 (d, J = 7.2 Hz, 3H) 252 (R or
S)-(.+-.)-1-(1- NMR (400 MHz, CDCl.sub.3): 379
cyclopropylethyl)-N-((4,6- .delta. 8.32-8.34 (d, 1H), 8.18-8.2 (d,
1H), dimethyl-2-oxo-1,2- 7.27-7.30 (m, 1H), 6.70 (s, 1H), 4.47 (s,
dihydropyridin-3-yl)methyl)- 2H), 3.94-3.95 (d, 1H), 2.61 (s, 3H),
2-methyl-1H-pyrrolo[2,3- 2.43 (s, 3H), 2.29-2.30 (s, 3H),
b]pyridine-3-carboxamide 1.57-1.59 (d, 3H), 0.63-0.64 (t, 1H), PEAK
1 0.27-0.64 (m, 2H), 0.02-0.04 (t, 1H) 253 (R or S)-(.+-.)-1-(1-
NMR (400 MHz, CDCl.sub.3): 379 cyclopropylethyl)-N-((4,6- .delta.
8.32-8.34 (d, 1H), 8.18-8.2 (d, 1H), dimethyl-2-oxo-1,2- 7.27-7.30
(m, 1H), 6.70 (s, 1H), 4.47 (s, dihydropyridin-3-yl)methyl)- 2H),
3.94-3.95 (d, 1H), 2.61 (s, 3H), 2-methyl-1H-pyrrolo[2,3- 2.43 (s,
3H), 2.29-2.30 (s, 3H), b]pyridine-3-carboxamide 1.57-1.59 (d, 3H),
0.63-0.64 (t, 1H), PEAK 2 0.27-0.64 (m, 2H), 0.02-0.04 (t, 1H) 256
(R or S)-N-((4-methoxy-6- (400 MHz, CDCl.sub.3) .delta. 13.23 (s,
1H), 399 methyl-2-oxo-1,2- 8.16-8.17 (m, 1H), 8.11-8.13 (m, 1H),
dihydropyridin-3-yl)methyl)- 7.57-7.60 (t, J = 5.2 Hz, 1H),
1-(1-methoxypropan-2-yl)-2- 6.93-6.96 (m, 1H), 5.92 (s, 1H),
4.82-4.83 (d, J = 2.4 Hz, methyl-1H-pyrrolo[2,3- 1H), 4.65-4.66 (d,
J = 6.4 Hz, b]pyridine-3-carboxamide 2H), 3.89 (s, 3H), 3.81-3.85
(m, 1H), PEAK 1 3.22 (s, 3H), 2.79 (s, 3H), 2.17 (s, 3H), 1.64-1.66
(d, J = 8.0 Hz, 3H) 257 (R or S)-N-((4-methoxy-6- (400 MHz,
CDCl.sub.3) .delta. 13.23 (s, 1H), 399 methyl-2-oxo-1,2- 8.16-8.17
(m, 1H), 8.11-8.13 (m, 1H), dihydropyridin-3-yl)methyl)- 7.57-7.60
(t, J = 5.2 Hz, 1H), 1-(1-methoxypropan-2-yl)-2- 6.93-6.96 (m, 1H),
5.92 (s, 1H), 4.82-4.83 (d, J = 2.4 Hz, methyl-1H-pyrrolo[2,3- 1H),
4.65-4.66 (d, J = 6.4 Hz, b]pyridine-3-carboxamide 2H), 3.89 (s,
3H), 3.81-3.85 (m, 1H), PEAK 2 3.22 (s, 3H), 2.79 (s, 3H), 2.17 (s,
3H), 1.64-1.66 (d, J = 8.0 Hz, 3H) 307 Trans-(R or S, R or
S)--N-((4- (CDCl.sub.3, 400 MHz) .delta. 7.85 (t, J = 6.4 Hz, 412
methoxy-6-methyl-2-oxo-1,2- 1H), 7.45 (s, 2H), 7.08-7.03 (m,
dihydropyridin-3-yl)methyl)- 2H), 5.93 (s, 1H), 4.71-4.61 (m, 2H),
1-(3-methoxybutan-2-yl)-2- 4.36 (s, 1H), 3.90 (s, 4H), 2.95 (s,
3H), methyl-1H-indole-3- 2.75 (s, 3H), 2.17 (s, 3H), 1.57 (d, J =
7.2 Hz, carboxamide 3H), 1.23 (d, J = 6.0 Hz, 3H) PEAK 1 308
Trans-(R or S, R or S)--N-((4- (CDCl.sub.3, 400 MHz) .delta. 7.85
(t, J = 6.4 Hz, 412 methoxy-6-methyl-2-oxo-1,2- 1H), 7.45 (s, 2H),
7.08-7.03 (m, dihydropyridin-3-yl)methyl)- 2H), 5.93 (s, 1H),
4.71-4.61 (m, 2H), 1-(3-methoxybutan-2-yl)-2- 4.36 (s, 1H), 3.90
(s, 4H), 2.95 (s, 3H), methyl-1H-indole-3- 2.75 (s, 3H), 2.17 (s,
3H), 1.57 (d, J = 7.2 Hz, carboxamide 3H), 1.23 (d, J = 6.0 Hz, 3H)
PEAK 2
Example 1
Synthesis of tert-butyl
1-(2,3-dihydro-1H-inden-1-yl)-2-methyl-1H-pyrrolo[2,3-b]pyridine-3-carbox-
ylate
[0120] The title compound as starting material in Step 3 of Example
36 in the synthesis of certain compounds of the invention.
Tert-butyl 2-methyl-1H-pyrrolo[2,3-b]pyridine-3-carboxylate
##STR00071##
[0121] To a 500 mL round-bottom flask that contains
N-acetyl-N-(3-bromopyridin-2-yl)acetamide (14.815 g, 57.6 mmol),
was added copper(I) iodide (1.098 g, 5.76 mmol), L-proline (1.327
g, 11.53 mmol), cesium carbonate (28.2 g, 86 mmol), then t-butyl
acetoacetate (11.47 ml, 69.2 mmol) and dioxane (100 mL). The
reaction was vac/purged with N.sub.2 3.times. then fitted with a
septum and a N.sub.2 inlet and heated overnight at 70.degree. C.
The inorganic solids were removed by filtration over celite and the
cake was washed with 100 mL EtOAc. This solution was concentrated
and the residue was partitioned between 250 mL brine and 250 mL
EtOAc. The aq. Layer was further extracted with EtOAc (2.times.250
mL) and the combined organic layer was dried over Na.sub.2SO.sub.4,
filtered, concentrated and purified by CC using 1:1 EtOAc:Hex as
eluent to provide (2.7 g, 20.2%) of tert-butyl
2-methyl-1H-pyrrolo[2,3-b]pyridine-3-carboxylate. LRMS (M+H.sup.+)
m/z: calc'd 233.28. found 233.1.
Tert-butyl
1-(2,3-dihydro-1H-inden-1-yl)-2-methyl-1H-pyrrolo[2,3-b]pyridin-
e-3-carboxylate
##STR00072##
[0122] A solution of ethyl tert-butyl
2-methyl-1H-pyrrolo[2,3-b]pyridine-3-carboxylate (100 mg, 0.74
mmol), 2,3-dihydro-1H-inden-1-ol (176 mg, 0.74 mmol), PPh.sub.3
(195 mg, 1.49 mmol) was stirred in dry THF (10 mL) at 0.degree. C.
under a nitrogen atmosphere. To this mixture was added drop-wise
DIAD (150 mg, 1.48 mmol) over a period of 5 min, and the reaction
was stirred at room temperature for 16 hours. The mixture was
washed with brine, dried and concentrated to afford the crude
product. The crude product was purified by silica gel
chromatography (petroleum ether/ethyl acetate=5:1) to afford the
tert-butyl-1-(2,3-dihydro-1H-inden-1-yl)-2-methyl-1H-pyrrolo[2,3-b]pyridi-
ne-3-carboxylate (150 mg, 60%).
[0123] The compound shown in the following table was prepared
according to the general procedure outlined above using the
appropriate starting materials and modifications.
TABLE-US-00021 Name Structure m/z (.+-.)-tert-butyl 1-(1-
cyclopropylethyl)-2-methyl- 1H-pyrrolo[2,3-b]pyridine-
3-carboxylate ##STR00073## 301
[0124] Each of the above alkyl carboxylates was used as starting
material in Step 3 of Example 1 in the synthesis of certain
compounds of the invention.
Example 13
Synthesis of isolated
N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-1-((2R
or 2S, 3R or
3S)-3-methoxybutan-2-yl)-2-methyl-1H-pyrrolo[2,3-b]pyridine-3-c-
arboxamide diastereomers (Compounds 261, 266, 267 and 302)
Step 1: Tert-butyl
2-methyl-1-(3-oxobutan-2-yl)-1H-pyrrolo[2,3-b]pyridine-3-carboxylate
##STR00074##
[0125] To a solution of tert-butyl
2-methyl-1H-pyrrolo[2,3-b]pyridine-3-carboxylate (5.0 g, 21.53
mmol) in CH.sub.3CN (50 mL) was added Cs.sub.2CO.sub.3 (21.0 g,
64.58 mmol), potassium iodide (3.57 g, 21.53 mmol). The mixture was
stirred at 27.degree. C. for 30 minutes. Then 3-chlorobutan-2-one
(2.75 g, 25.83 mmol) was added and the mixture was stirred at
70.degree. C. for 12 hours. The mixture was filtered and the
filtrate was concentrated. The residue was purified by column
(Elute:Petroleum ether:Ethyl acetate 50:1) to give tert-butyl
2-methyl-1-(3-oxobutan-2-yl)-1H-pyrrolo[2,3-b]pyridine-3-carboxylate
as a yellow-green oil. (3.23 g, yield 50%) LCMS (M+H.sup.+) m/z:
calcd 303.37. found 302.9. 1H NMR (400 MHz, CDCl.sub.3): a
8.32-8.30 (m, 1H), 8.25-8.23 (m, 1H), 7.17-7.14 (m, 1H), 5.50-5.44
(m, 1H), 2.71 (s, 3H), 1.96 (s, 3H), 1.65-1.67 (d, 3H), 1.64 (s,
9H).
Step 2: Tert-butyl
1-(3-hydroxybutan-2-yl)-2-methyl-1H-pyrrolo[2,3-b]pyridine-3-carboxylate
##STR00075##
[0126] To the solution of tert-butyl
2-methyl-1-(3-oxobutan-2-yl)-1H-pyrrolo[2,3-b]pyridine-3-carboxylate
(3.1 g, 10.25 mmol) in methanol (30 mL) was added sodium
borohydride (0.30 g, 8.2 mmol) at 0.degree. C. After 30 minutes,
another batch of sodium borohydride (0.30 g, 8.2 mmol) was added at
0.degree. C. After the reaction completed about 2 h later, water
(30 ml) was added dropwise very carefully to quench the reaction.
The mixture was extracted with CH.sub.2Cl.sub.2. The extraction was
dried over Na.sub.2SO.sub.4, filtered and concentrated under vacuum
to give tert-butyl
1-(3-hydroxybutan-2-yl)-2-methyl-1H-pyrrolo[2,3-b]pyridine-3-carboxylate
as a yellow solid. (3.0 g, yield 96%) LCMS (M+H.sup.+) m/z: calcd
305.38. found 304.9. 1H NMR (400 MHz, CDCl.sub.3): a 8.31-8.29 (m,
1H), 8.13-8.12 (m, 1H), 7.11-7.07 (m, 1H), 4.46-4.43 (m, 1H), 4.12
(m, 1H), 2.73 (s, 3H), 1.58 (s, 9H), 1.51-1.49 (d, 3H), 0.92-0.91
(d, 3H).
Step 3:
Tert-butyl-1-(3-methoxybutan-2-yl)-2-methyl-1H-pyrrolo[2,3-b]pyrid-
ine-3-carboxylate
##STR00076##
[0127] To dry THF (20 mL) was added NaH (60% in mineral oil, 2.37
g, 59.14 mmol). Then the mixture was stirred at 27.degree. C. for
20 minutes, then tert-butyl
1-(3-hydroxybutan-2-yl)-2-methyl-1H-pyrrolo[2,3-b]pyridine-3-carboxylate
(3.0 g, 9.86 mmol) was added. The mixture was stirred at 27.degree.
C. for 1 hour, then added by CH.sub.3I (13.99 g, 98.6 mmol). The
mixture was stirred for 12 hours at 27.degree. C. and then cooled
to 0.degree. C. Sat. NH.sub.4Cl was added and extracted with
CH.sub.2Cl.sub.2. The extraction was dried over sodium sulfate,
filtered and concentrated to give
tert-butyl-1-(3-methoxybutan-2-yl)-2-methyl-1H-pyrrolo[2,3-b]pyridin-
e-3-carboxylate as a yellow oil. (3.2 g, yield 100%) LCMS
(M+H.sup.+) m/z: calcd. 319.41. found 318.9.
Step 4:
1-(3-methoxybutan-2-yl)-2-methyl-1H-pyrrolo[2,3-b]pyridine-3-carbo-
xylic acid
##STR00077##
[0128] To the pre-cooled solution of tert-butyl
1-(3-methoxybutan-2-yl)-2-methyl-1H-pyrrolo[2,3-b]pyridine-3-carboxylate
(3.0 g, 9.42 mmol) in CH.sub.2Cl.sub.2 (20 mL) was added
trifluoroacetic acid (20 mL) dropwise. The solution was stirred at
27.degree. C. for 1.5 hours. The solvent was removed under vacuum
at 27.degree. C. The residue was used for next step without
purified. LCMS (M+H+) m/z: calcd 263.30. found 262.9.
Step 5:
N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-1-(3--
methoxybutan-2-yl)-2-methyl-1H-pyrrolo[2,3-b]pyridine-3-carboxamide
##STR00078##
[0129] To a solution of
1-(3-methoxybutan-2-yl)-2-methyl-1H-pyrrolo[2,3-b]pyridine-3-carboxylic
acid (2.4 g, 9.15 mmol) in DMF (30 mL) was added TEA (4.2 g, 41.50
mmol), 3-(aminomethyl)-4-methoxy-6-methylpyridin-2(1H)-one
hydrochloride (2.1 g, 12.81 mmol) After stirred for 10 minutes at
27.degree. C., the mixture was cooled and added HATU (5.56 g, 14.64
mmol). The mixture was stirred at 27.degree. C. for 72 hours and
30% of S.M. remained. Then the mixture was heated at 80.degree. C.
for 5 hours. The solution was diluted with brine (100 mL) and
extracted with CH.sub.2Cl.sub.2 (100 mL*3). The extractions were
combined and dried over Na.sub.2SO.sub.4. The solvent was
evaporated under vacuum and the residue was purified by flash
column (Eluent:dichloromethane:methanol 95:5) to give
N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-1-(3-methoxy-
butan-2-yl)-2-methyl-1H-pyrrolo[2,3-b]pyridine-3-carboxamide. (3.6
g, yield 95%)
Step 6: Separation of
N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-1-(3-methoxy-
butan-2-yl)-2-methyl-1H-pyrrolo[2,3-b]pyridine-3-carboxamide:
Isomers (Compounds 261, 266, 267, and 302)
##STR00079## ##STR00080##
[0130] The mixture of isomers from Step 5,
N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-1-(3-methoxy-
butan-2-yl)-2-methyl-1H-pyrrolo[2,3-b]pyridine-3-carboxamide was
purified by prep-HPLC (Condition: Column: SHIMADZU LC-8A, 250*50
mm*10 um; Mobile phase A: water with 0.2% formic acid; Mobile phase
B: MeCN; column temperature: 30.degree. C.; Gradient: B in A
10-50%) to give a major isomer pair (Compound 261 and Compound 266
combined) (1.0 g, purity 98.8%) and a minor isomer pair (Compound
267 and Compound 302 combined) (180 mg, purity 63%). The resulting
isomer pairs were individually separated by SFC (Condition: Column:
Chiralpak AD 250*30 mm*5 um; Mobile phase A: Supercritical
CO.sub.2; Mobile phase B: IPA+NH.sub.3.H.sub.2O; Gradient: B/A:
75:25) to give the following individual single compounds:
[0131] Compound 261,
N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-1-((2R
or 2S, 3R or
3S)-3-methoxybutan-2-yl)-2-methyl-1H-pyrrolo[2,3-b]pyridine-3-c-
arboxamide (Major Isomer Pair; Peak 1): .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta. 8.173-8.157 (m, 1H), 8.140-8.116 (m, 1H),
7.582-7.555 (m, 1H), 6.968-6.936 (m, 1H), 5.927 (s, 1H),
4.707-4.609 (m, 2H), 4.348 (s, 1H), 3.892 (s, 3H), 2.869 (s, 3H),
2.788 (s, 3H), 2.173 (s, 3H), 1.644-1.627 (d, 3H), 1.263-1.249 (d,
3H).
[0132] Compound 266,
N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-1-((2R
or 2S, 3R or
3S)-3-methoxybutan-2-yl)-2-methyl-1H-pyrrolo[2,3-b]pyridine-3-c-
arboxamide (Major Isomer Pair; Peak 2): .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta. 8.179-8.163 (m, 1H), 8.143-8.120 (m, 1H),
7.558-7.531 (m, 1H), 6.986-6.954 (m, 1H), 5.931 (s, 1H),
4.702-4.605 (m, 2H), 3.897 (s, 3H), 2.892 (s, 3H), 2.789 (s, 3H),
2.189 (s, 3H), 1.647-1.629 (d, 3H), 1.267-1.252 (d, 3H).
[0133] Compound 267,
N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-1-((2R
or 2S, 3R or
3S)-3-methoxybutan-2-yl)-2-methyl-1H-pyrrolo[2,3-b]pyridine-3-c-
arboxamide (Minor Isomer Pair; Peak 1): .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta. 8.174-8.162 (d, 1H), 8.111-8.094 (d, 1H),
7.551-7.526 (m, 1H), 6.993-6.961 (m, 1H), 5.935 (s, 1H),
4.683-4.579 (m, 2H), 3.887 (s, 3H), 3.442 (s, 3H), 2.753 (s, 3H),
2.194 (s, 3H), 1.695-1.678 (d, 3H), 0.781-0.768 (d, 3H).
[0134] Compound 302,
N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-1-((2R
or 2S, 3R or
3S)-3-methoxybutan-2-yl)-2-methyl-1H-pyrrolo[2,3-b]pyridine-3-c-
arboxamide (Minor Isomer Pair; Peak 2): .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta. 8.177-8.166 (d, 1H), 8.122-8.104 (d, 1H),
7.587-7.562 (m, 1H), 6.984-6.952 (m, 1H), 5.933 (s, 1H),
4.698-4.591 (m, 2H), 4.426 (s, 2H), 3.983 (s, 3H), 3.448 (s, 3H),
2.764 (s, 3H), 2.180 (s, 3H), 1.701-1.684 (d, 3H), 0.786-0.772 (d,
3H).
Example 14
Synthesis of
(.+-.)-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methyl-1--
(1-phenylethyl)-1H-pyrrolo[2,3-c]pyridine-3-carboxamide
Step 1: 1-(3-methoxyphenyl)ethanol
##STR00081##
[0135] To a stirred solution of 3-Amino-4-picoline (7 g, 64.8 mmol)
in anhydrous THF (200 mL), sec-BuLi (150 mL, 1.3M in cyclohexane,
194 mmol) was added dropwise over 20 minutes at -78.degree. C. The
solution was warmed to room temperature and stirred at 3 hours.
Ethyl acetate (2.3 g, 25.9 mmol) was added dropwise into the
reaction at -78.degree. C. and the mixture was stirred at the same
temperature for 2 hours. Methanol (50 mL) was added dropwise into
the reaction over 10 minutes. The mixture was warmed to room
temperature and stirred for 1 hour. A half-saturated NH4Cl (250 mL)
was added. The mixture was extracted with EA. The combined organic
layers were washed with brine, dried and concentrated to afford the
crude product. The crude product was purified by silica gel
chromatography (petroleum ether/ethyl acetate=10:1) to afford
2-methyl-1H-pyrrolo[2,3-c]pyridine (2.5 g, 73.5%).
Step 2:
2,2,2-trichloro-1-(2-methyl-1H-pyrrolo[2,3-c]pyridin-3-yl)ethanone
##STR00082##
[0136] To a stirred solution of 2-methyl-1H-pyrrolo[2,3-c]pyridine
(2.5 g, 18.9 mmol) and aluminum chloride (5 g, 37.8 mmol) in DCM
(100 mL), trichloroacetylchloride (4.1 g, 22.7 mmol) was added
dropwise into the reaction over 0.5 hours at room temperature.
After stirring 2 hours, the reaction was cooled to 0.degree. C. and
was quenched with water (100 mL). The resulting precipitate was
isolated by filtration to afford
2,2,2-trichloro-1-(2-methyl-1H-pyrrolo[2,3-c]pyridin-3-yl)ethanone
which was used for next step without further purification. Assumed
100% yield. (5.24 g).
Step 3: Methyl 2-methyl-1H-pyrrolo[2,3-c]pyridine-3-carboxylate
##STR00083##
[0137] A mixture of
2,2,2-trichloro-1-(2-methyl-1H-pyrrolo[2,3-c]pyridin-3-yl)ethanone
(5.24 g, 18.9 mmol) and KOH (1.2 g, 20.9 mmol) in MeOH (100 mL) was
stirred at room temperature for 16 hour. The reaction mixture was
concentrated to remove MeOH, the residue was partitioned between EA
and Water. The organic layer was washed with brine, dried and
concentrated to afford methyl
2-methyl-1H-pyrrolo[2,3-c]pyridine-3-carboxylate (3 g, 83%).
Step 4: Methyl methyl
2-methyl-1-(1-phenylethyl)-1H-pyrrolo[2,3-c]pyridine-3-carboxylate
##STR00084##
[0138] A mixture of methyl
2-methyl-1H-pyrrolo[2,3-c]pyridine-3-carboxylate (550 mg, 2.89
mmol) and sodium hydride (200 mg, 4.34 mmol) in
N,N-dimethylformamide (3.0 mL) was stirred at room temperature for
0.5 hour, and then (1-bromoethyl)benzene (589 mg, 3.18 mmol) was
added. The mixture was stirred at room temperature for 3 hours. The
reaction mixture was poured into saturated NH.sub.4Cl and extracted
with ethyl acetate. Organic layers were combined and concentrated
to give a residue. The residue was purified by chromatography
(petroleum ether/ethyl acetate=5:1) to give methyl
2-methyl-1-(1-phenylethyl)-1H-pyrrolo[2,3-c]pyridine-3-carboxylate
(800 mg, 94%).
Step 5:
2-methyl-1-(1-phenylethyl)-1H-pyrrolo[2,3-c]pyridine-3-carboxylic
acid
##STR00085##
[0139] To a mixture of methyl
2-methyl-1-(1-phenylethyl)-1H-pyrrolo[2,3-c]pyridine-3-carboxylate
(800 mg, 2.72 mmol) and KOH (1.5 g, 27.2 mmol) in (15 mL) and water
(5 mL) was refluxed for 2 hours. The mixture was adjust PH to 2 by
10% HCl and extracted with EA. The combined organic layers were
washed with brine, dried and concentrated to afford the crude
product. The crude product was used into the next step without more
purification. 100% yield. (760 mg).
Step 6:
(.+-.)-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-me-
thyl-1-(1-phenylethyl)-1H-pyrrolo[2,3-c]pyridine-3-carboxamide
(Compound 203)
##STR00086##
[0140] A mixture of
2-methyl-1-(1-phenylethyl)-1H-pyrrolo[2,3-c]pyridine-3-carboxylic
acid (280 mg, 1.0 mmol) was added HATU (456 mg, 1.2 mmol), TEA (1
g, 10 mmol) and 3-(aminomethyl)-4,6-dimethylpyridin-2(1H)-one (182
mg, 1.2 mmol) in anhydrous dichloromethane (30 mL) was stirred at
room temperature for 16 hours. To the reaction mixture was added
water (10 mL), extracted with dichloromethane (30 mL.times.2). The
organic layers were combined and concentrated to give a residue.
The residue was rereystallized from MeCN to afford compound
N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methyl-1-(1-phen-
ylethyl)-1H-pyrrolo[2,3-c]pyridine-3-carboxamide as an off-white
solid (80 mg, 21.6%). LRMS (M+H) m/z: calcd 414.21. found 414.
.sup.1H NMR (400 MHz, Methanol-d4) .delta.: 8.84 (s, 1H), 8.16 (d,
J=7.6 Hz, 1H), 8.03 (d, J=6.8 Hz, 1H), 7.44-7.37 (m, 5H), 6.09 (s,
1H), 6.01-5.99 (m, 1H), 4.49 (s, 2H), 2.73 (s, 3H), 2.38 (s, 3H),
2.22 (s, 3H), 2.06 (d, J=7.2 Hz, 3H).
[0141] The compounds shown in the following table were prepared
according to the general procedure outlined in this example using
the appropriate starting materials and modifications. Structures
are shown in FIG. 1.
TABLE-US-00022 Compound Name NMR m/z 240 (.+-.)-N-((4,6-dimethyl-2-
(400 MHz, CHLOROFORM-d) .delta. ppm 383 oxo-1,2-dihydropyridin-
1.63 (br. s., 3 H) 2.21 (s, 3 H) 2.41 (s, 3 H) 2.73 (s,
3-yl)methyl)-1-(1- 3 H) 3.24 (s, 3 H) 2.72 (dd, J = 9.81, 5.40 Hz,
methoxypropan-2-yl)-2- 1 H) 3.80-3.88 (m, 1 H) 4.60 (d, J = 5.95
Hz, methyl-1H-pyrrolo[2,3- 2 H) 4.71 (dd, J = 13.23, 7.06 Hz, 1 H)
5.92 (s, c]pyridine-3- 1 H) 7.31 (d, J = 5.73 Hz, 1 H) 7.38 (br.
s., 1 carboxamide H) 8.26 (d, J = 5.29 Hz, 1 H) 9.09 (br. s., 1 H)
11.07 (br. s., 1 H) 243 (.+-.)-N-((4-methoxy-6- (400 MHz,
CHLOROFORM-d) .delta. ppm 431 methyl-2-oxo-1,2- 1.62 (br. s., 3 H)
2.26 (s, 3 H) 2.75 (s, 3 H) 3.25 (s, dihydropyridin-3- 3 H) 3.72
(dd, J = 9.81, 5.40 Hz, 1 H) yl)methyl)-2-methyl-1- 3.80-3.87 (m, 1
H) 3.90 (s, 3 H) 4.65 (d, J = 5.29 Hz, (1-phenylethyl)-1H- 2 H)
4.71 (dd, J = 13.78, 6.95 Hz, 1 H) pyrrolo[2,3-c]pyridine- 5.93 (s,
1 H) 7.32 (br. s., 1 H) 7.50 (br. s., 1 3-carboxamide H) 8.25 (br.
s., 1 H) 9.11 (br. s., 1 H)
Example 15
General Procedures for Synthesizing Other Compounds of the
Invention
General Procedure A
Indole Alkyation
##STR00087##
[0142] To a cooled (0.degree. C.) solution of NH indole ester (1
equivalent) in N,N-dimethylformamide (volume to make concentration
0.4M) was added sodium hydride (60% w/w, 1.1 equivalents relative
to indole). The resultant mixture was stirred for 15 minutes. Then
RX (2 equivalents) was added and the reaction was allowed to warm
to room temperature. The reaction was maintained at ambient
temperature for 12 hours. The reaction mixture was poured into
saturated ammonium chloride solution (100 mL) with stirring. The
mixture was extracted with ethyl acetate (200 mL.times.2) and the
combined organic phase was washed with brine, dried over magnesium
sulfate, filtered, and concentrated to give crude product which was
purified by column chromatography (silica gel, petroleum
ether/ethyl acetate=20:1) to afford the desired alkylated Indole
ester product.
General Procedure B
Saponification of Alkylated Indole Ester
##STR00088##
[0143] To a solution of alkylated Indole ester (1 equivalent) in
tetrahydrofuran:methanol:water (2.5:5:1, volume to make
concentration 0.05M) was added lithium hydroxide (4 equivalents).
The resultant reaction mixture was stirred at 60.degree. C. for 48
hours. The mixture was concentrated in vacuo. Then the residue was
diluted with water (40 mL) and slowly acidified with 1N hydrogen
chloride to pH=4-5. The mixture was extracted with ethyl acetate
(100 mL.times.3). The combined organic layers were washed with
brine, dried over magnesium sulfate, filtered and concentrated to
give crude indole acid, which was used in the subsequent step
without additional purification.
General Procedure C
Amide Bond Formation
##STR00089##
[0144] To a solution of Indole acid (1 equivalent) in
dichloromethane (volume to make concentration 0.05M) were added
1-hydroxybenzotriazole (1.5 equivalents),
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.5
equiv.) and triethylamine (3 equiv.). The resultant mixture was
stirred at room temperature for 30 minutes. Then Pyridone amine
(1.2 equiv.) was added and the resultant mixture was stirred at
room temperature for 16 hours. Water (50 mL) was added to the
mixture. The mixture was extracted with dichloromethane (100
mL.times.2). The organic layer was concentrated in vacuo to provide
crude product which was purified by column chromatography (silica
gel, dichloromethane/methanol=20:1) to afford the target
compound.
General Procedure D
Chiral Chromatography
[0145] Separation of chiral compounds was accomplished via normal
phase HPLC or SFC (supercritical carbon dioxide fluid
chromatography). Separated compounds were typically >95% ee. The
absolute configuration of chiral centers was not determined.
General Procedure L
Sulphonylation
##STR00090##
[0146] To a solution of Chiral amine (1 equiv.) in dichloromethane
(volume to make concentration 0.1M) was added triethylamine (4
equiv.) at 18.degree. C. under N.sub.2. The reaction was cooled to
0.degree. C. and methanesulfonyl chloride (1.5 equiv.) was added.
The reaction was stirred at 0.degree. C. for 1 h. Then the mixture
was concentrated in vacuo and methanol and potassium carbonate were
added and the reaction was stirred for another 1 h. The mixture was
filtered and the crude product was purified by
preparative-HPLC.
[0147] The table below lists compounds of the invention and which
of the above general methods was used in their synthesis.
Structures of these compounds are set forth in FIG. 1.
TABLE-US-00023 General Methods Compound Used and Notes Name NMR
data m/z 370 General .sup.1H NMR (CDCl3, 400 MHz) 517 procedure L
on .delta. 12.12 (s, 1H), 9.20 (s, 1H), (.+-.)-N-((4- 8.78 (s, 1H),
7.68 (s, 1H), methoxy-6- 5.97 (s, 1H), 4.70-4.58 (m, methyl-2-oxo-
2H), 3.91 (s, 4H), 3.62 (d, J = 12.0 Hz, 1,2- 1H), 2.76-2.67 (m,
dihydropyridin- 7H), 2.45 (dd, J.sub.1 = 2.0 Hz, J.sub.2 = 11.6 Hz,
3-yl)methyl)-6- 1H), 2.32 (s, 3H), methyl-7-(1- 2.10 (t, J = 12.0
Hz, 1H), (piperidin-4- 1.67 (d, J = 6.8 Hz, 4H), yl)ethyl)-7H-
1.48-1.40 (m, 1H), pyrrolo[2,3- 1.37-1.29 (m, 1H), 1.27-1.19 (m,
1H), d]pyrimidine-5- 0.9-0.78 (m, 1H). carboxamide
Example 16
IC.sub.50 Measurements for Inhibitors Using EZH2
[0148] EZH2 Assay:
[0149] Assays were carried out by mixing rPRC2 together with
biotinylated oligonucleosome substrates in the presence of the
radio-labeled enzyme co-factor, S-adenosyl-L-methionine (.sup.3H
SAM) (Perkin Elmer) and monitoring the enzymatically mediated
transfer of tritiated methyl groups from .sup.3H SAM to histone
lysine residues. The amount of resulting tritiated methyl histone
product was measured by first capturing the biotinylated
oligonucleosomes in streptavidin (SAV) coated FlashPlates (Perkin
Elmer), followed by a wash step to remove un-reacted .sup.3H SAM,
and then counting on a TopCount NXT 384 well plate scintillation
counter (Perkin Elmer). The final assay conditions for EZH2 were as
follows: 50 mM Tris Buffer pH 8.5, 1 mM DTT, 69 .mu.M Brij-35
detergent, 5.0 mM MgCl.sub.2, 0.1 mg/mL BSA, 0.2 .mu.M .sup.3H SAM,
0.2 .mu.M biotinylated oligonucleosomes, 3.6 .mu.M H3K27me3 peptide
and 2 nM EZH2.
[0150] Compound IC.sub.50 measurements were obtained as follows:
Compounds were first dissolved in 100% DMSO as 10 mM stock
solutions. Ten point dose response curves were generated by
dispensing varying amounts of the 10 mM compound solution in 10
wells of the 384 well plate (Echo; Labcyte), pure DMSO was then
used to backfill the wells to insure all wells have the same amount
of DMSO. A 12.5 .mu.L volume of the HMT enzyme, H3K27me3 peptide
and oligonucleosome substrate in assay buffer was added to each
well of the assay plate using a Multidrop Combi (ThermoFisher).
Compounds were pre-incubated with the enzyme for 20 min, followed
by initiation of the methyltransferase reaction by addition of 12.5
.mu.L of .sup.3H SAM in assay buffer (final volume=25 .mu.L). The
final concentrations of compounds ranged from a top default
concentration of 80 .mu.M down to 0.16 .mu.M in ten 2-fold dilution
steps. Reactions were carried out for 60 minutes and quenched with
20 .mu.L per well of 1.96 mM SAH, 50 mM Tris pH 8.5, 200 mM EDTA.
Stopped reactions were transferred to SAV coated Flashplates
(Perkin Elmer), incubated for 120 min, washed with a plate washer,
and then read on the TopCount NXT (1.0 min/well) to measure the
amount of methyl histone product formed during the reaction. The
amount of methyl histone product was compared with the amount of
product formed in the 0% and 100% inhibition control wells allowing
the calculation of % Inhibition in the presence of the individual
compounds at various concentrations. IC.sub.50's were computed
using a 4 parameter fit non-linear curve fitting software package
(XLFIT, part of the database package, ActivityBase (IDBS)) where
the four parameters were IC.sub.50, Hill slope, pre-transitional
baseline (0% INH), and post-transitional baseline (100% INH); with
the latter two parameters being fixed to zero and 100%,
respectively, by default.
[0151] Assay for Y641N EZH2 was performed as above using
reconstituted H3K27Me2 oligonucleosomes as substrate.
[0152] Table 2 shows the activity of selected compounds of this
invention in the EZH2 and Y641N EZH2 activity inhibition assay.
IC.sub.50 values are reported as follows: "A" indicates an
IC.sub.50 value of less than 100 nM; "B" indicates an IC.sub.50
value of 100 nM to 1 .mu.M; "C" indicates an IC.sub.50 value of
greater than 1 .mu.M and less than 10 .mu.M for each enzyme; "D"
indicates an IC.sub.50 value of greater than 10 .mu.M for each
enzyme; and "*(X .mu.M)" indicates that no inhibition was observed
at the highest concentration (i.e., X .mu.M) of compound
tested.
TABLE-US-00024 TABLE 2 IC50 Values for Compounds of Formula I
against EZH2 and Y641N EZH2 Mutant Enzymes. Compound Y641N No. EZH2
IC.sub.50 EZH2 IC.sub.50 183 A A 204 A B 211 A B 212 A B 217 B B
218 A A 219 A A 223 A B 224 A A 229 C D 230 A B 231 A B 234 C D 235
B C 236 *(0.5 .mu.M) *(10 .mu.M) 240 A B 241 A B 243 A B 252 A B
253 A B 256 A B 257 B C 261 A A 266 B B 267 A B 273 A A 275 A A 277
A A 280 B C 284 A B 288 A A 290 A B 293 A B 294 A A 298 A A 299 A A
300 A A 302 B C 304 A A 306 A A 307 A A 308 B B 310 A A 313 A A 314
A A 316 A A 317 A A 321 A A 327 A A 335 A A 336 A A 337 A A 341 A A
342 A A 343 A A 344 A A 345 A A 346 A A 347 A A 352 A A 355 A A 356
A A 357 A A 358 A A 359 A A 360 A A 362 A A 363 A A 364 A B 365 A A
366 A A 367 A A 368 A A 369 A A 370 A A 373 A A 375 A A 376 A A 377
A A
Example 17
EC50 Measurements for Inhibitors in HeLa Cell Assays
[0153] H3K27me3 MSD Hela Assay.
[0154] Trypsinized HeLa cells were counted and diluted in 10% DMEM
(Life Technologies, Cat. #10569) to 5000 cells/75 .mu.L.
Seventy-five .mu.L of cells were place in each well of a 96-well
flat-bottomed plate and incubated at 37.degree. C. for 4 hours.
Twenty-five .mu.L of test compound (at various concentrations) was
added to the cells and incubation continued at 37.degree. C. for 96
hours. Media was then removed and the cells rinsed once with ice
cold PBS. Forty .mu.L of ice-cold MSD Buffer AT (10 mM HEPES, pH
7.9, 5 mM MgCl.sub.2, 0.25M sucrose, Benzonase (1:10000), 1% Triton
X-100 supplemented with fresh 1.times. Protease Inhibitor cocktail
and 1 mM 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride
(AEBSF)) was added to each well and the plates placed on ice for 30
minutes. Ten .mu.L of 5M NaCl was then added to each well and
incubation on ice continued for another 15 minutes. The material in
each well was suspended pipetting up and down and then transferred
to a new 96 well plate. The emptied wells were rinsed with 150 uL
ice-cold 20 mM Tris pH 7.5, 1 mM EDTA, 1 mM EGTA, supplemented with
fresh 1.times. Protease Inhibitor cocktail and 1 mM AEBSF ("NO salt
NO detergent buffer) and transferred to the respective wells in the
new plate. Three hundred .mu.L of NO Salt NO detergent buffer was
then added to each well of lysates and the plates frozen at
-80.degree. C.
[0155] On the same day, an appropriate number of MSD standard bind
96-well plates were coated with 30 .mu.L/well of total H3 capture
antibody (Millipore, Cat # MAB3422) at 1 .mu.g/mL concentration in
PBS. The antibody solution was evenly distributed first by tapping
gently on the sides of the plates and then by shaking the plates
for a few minutes at 1000 rpm. Antibody coated plates were stored
at 4.degree. C. overnight.
[0156] The next day the lysates are thawed to RT. The antibody
coated MSD plates are washed 3.times. with TBS-T (Tris-buffered
saline (Fisher Scientific, Cat #BP2471-1)+0.2% Tween-20).
One-hundred fifty .mu.L of 5% Blocker A in TBS-T is added to each
well. The wells are covered and shaken on a shaker at RT for one
hour. The Blocker A step is repeated a second time. After removing
the blocker, 25 .mu.L of cell lysate is transferred into each
antibody coated well. The plates are shaken for 2 hours at RT, the
lysate removed and the plates again washed with Blocker A in TBS-T.
Twenty-five .mu.L of appropriate freshly prepared antibody mix
(including both primary and secondary antibodies) is added to each
well and the plates shaken for 1 hour at RT. The antibody mix used
was one (or both) of those indicated in the table below:
TABLE-US-00025 Anti-rabbit Concentration Primary detection 1%
blocker A Ab (.mu.g/mL) Ab (.mu.L) Ab (.mu.L) (.mu.L) H3K27me3 33
37.88 5.00 5000 H3 12 52.08 5.00 5000
Both H3 antibodies were obtained from Cell Signalling (Cat #s 4499
and 9733). The goat anti-rabbit antibody was obtained from
Meso-Scale Discovery (Cat #R32AB-1).
[0157] The antibody mix was then removed and the wells washed with
Blocker A. One hundred-fifty .mu.L of freshly prepared 1.times.MSD
Read Buffer (Meso-Scale Discovery; Cat #R927C-2) was then added to
each well and the plates read on a MSD Sector 2400 Plate
Reader.
[0158] Data was analyzed using Assay Assistant (Constellation
Pharmaceuticals In-house product) and Activity Base (IDBS Ltd,
Surrey, UK) template. Data files were imported to Assay Assistant
and assay conditions were specified. A unique Analysis ID was
created and the data files exported to Activity Base. An analysis
template was created on Activity Base to measure dose-dependent
inhibition of H3K27me3 mark and cell viability respectively.
Readout of DMSO wells were used to normalize the data. Resulting
curves were fitted using Activity base software Model 205 (IDBS
Ltd, Surrey, UK). The data was checked for quality, validated and
integrated in excel format using SARview (IDBS Ltd, Surrey,
UK).
[0159] H3K27me3 Alpha Hela Assay (AlphaLISA).
[0160] Ten different doses of each test compound (in a series of
3-fold dilutions) were plated in duplicate 384-well tissue culture
treated plates (Catalog #781080; Greiner Bio One; Monroe, N.C.).
Hela cells grown in culture were trypsinized and counted using a
Countess.RTM. cell counter (Catalog #C10281; Life Technologies,
Grand Island, N.Y.). Cell were diluted to 67,000 cells per mL in
10% DMEM (Catalog #10569-010 Life Technologies, Grand Island, N.Y.)
and 15 .mu.L (1,000 cells) were plated into each well using the
Biotek MicroFlo.TM. Select Dispenser (BioTek Instruments, Inc.
Vermont, USA),) of the 384-well plate. Plates were incubated at
37.degree. C./5% CO.sub.2 for 72 hrs. One of the duplicate plates
was processed for HeLa assay and the other for viability.
[0161] To the plate processed for AlphaLISA was added 5 .mu.L per
well Cell-Histone Lysis buffer (1.times.) (Catalog # AL009F1 Perkin
Elmer; Waltham, Mass.) and the plate was incubated at RT for 30
minutes on a plate shaker with low speed (Model#4625-Q Thermo
Scientific; Waltham, Mass.). Then, 10 .mu.L per well Histone
Extraction buffer (catalog # AL009F2; Perkin Elmer; Waltham, Mass.)
was added and the plate further incubated at RT for 20 min on plate
shaker with low speed. To each well was then added 10 .mu.L per
well of a 5.times. mix of anti-K27me3 acceptor beads plus
Biotinylated anti-Histone H3 (C-ter) Antibody (diluted to 3 nM
final) (Catalog #AL118 Perkin Elmer; Waltham, Mass.). Dilution of
the acceptor beads and then anti-Histone H3 was with 1.times.
Histone Detection buffer (Catalog # AL009F3 Perkin Elmer; Waltham,
Mass.) which was produced diluted from the 10.times. stock
provided. The plate was sealed with an aluminum plate sealer and
incubated at 23.degree. C. for 60 min. We then added 10 .mu.L
5.times. solution of Streptavidin Donor beads (Catalog #6760002
Perkin Elmer; Waltham, Mass.) (20 .mu.g/mL final in 1.times.
Histone Detection Buffer), sealed the plate with Aluminum plate
sealer and incubated at 23.degree. C. for 30 min. The plates were
then read using an EnVision-Alpha Reader (model #2104 Perkin Elmer;
Waltham, Mass.).
[0162] Cell viability was assayed by adding 15 .mu.L of Cell Titer
Glo ((Catalog #G7571 Promega Madison, Wis.) to each well with cells
with media. The plates were incubated foat RT for 15-20 minutes on
a plate shaker at low speed. The plates were then read using an
EnVision-Alpha Reader (model #2104 Perkin Elmer; Waltham,
Mass.).
[0163] Data from both assays was analyzed using Assay Assistant
(Constellation Pharmaceuticals In-house product) and Activity Base
(IDBS Ltd, Surrey, UK) template. Data files were imported to Assay
Assistant and assay conditions were specified. A unique Analysis ID
was created and the data files exported to Activity Base. An
analysis template was created on Activity Base to measure
dose-dependent inhibition of H3K27me3 mark and cell viability
respectively. Readout of DMSO wells were used to normalize the
data. Resulting curves were fitted using Activity base software
Model 205 (IDBS Ltd, Surrey, UK). The data was checked for quality,
validated and integrated in excel format using SARview (IDBS Ltd,
Surrey, UK).
[0164] Table 3 shows the activity of selected compounds of this
invention in the two different HeLa cell assays described above.
EC.sub.50 values are reported as follows: "A" indicates an
EC.sub.50 value of less than 400 nM; "B" indicates an EC.sub.50
value of 400 nM to 2 .mu.M; "C" indicates an EC.sub.50 value of
greater than 2 .mu.M and less than 10 .mu.M for each enzyme; "D"
indicates an EC.sub.50 value of greater than 10 .mu.M for each
enzyme; and "*(X .mu.M)" indicates that no inhibition was observed
at the highest concentration (i.e., X .mu.M) of compound
tested.
TABLE-US-00026 TABLE 3 Ec50 Values for Selected Compounds of the
Invention In Hela Cells Expressing H3k27 Mutant EZH2. Com- pound
H3K27me3_Alpha_HeLa No. (EC50) H3K27me3_MSD_HeLa_(EC50) 204 B 211 B
212 B 218 A 219 B 224 A A 230 B 240 C 241 B 243 C 253 A 256 B 261 A
A 273 A 284 B 288 A B 294 A A 298 A A 300 A A 304 A A 310 A A 313 A
A 314 A 315 D 316 B 317 A 321 A 327 A 335 A 336 A 337 A 341 A 342 A
343 B 344 A 345 A 346 A 347 B 352 B 355 A 356 A 357 A 358 B 359 B
360 C 362 A 363 A 364 *(3.33 .mu.M) 365 A 366 B 367 A 368 A 369 A
370 *(3.33 .mu.M) 373 A 374 A 375 A 376 NaN 377 B
Example 18
Tumor Growth Inhibition Analysis
[0165] The anti-tumor efficacy of Compound 362 and 365 in the
subcutaneous Karpas422 human lymphoma xenograft model in female
CB-17 SCID mice was as follows.
[0166] Animals
Species: Mus Musculus
[0167] Strain: CB-17 SCID mice Age: 6-8 weeks Sex: female Body
weight: 18-22 g Number of animals: 50 mice plus spare Animal
supplier: Shanghai SLAC Laboratory Animal Co., LTD.
[0168] Cell Culture
[0169] The Karpas422 tumor cells were maintained in vitro as
suspension culture in RPMI1640 medium supplemented with 10% heat
inactivated fetal calf serum at 37.degree. C. in an atmosphere of
5% CO.sub.2 in air. The tumor cells were routinely subcultured
twice weekly. The cells growing in an exponential growth phase were
harvested and counted for tumor inoculation.
[0170] Tumor Inoculation
[0171] Each mouse was inoculated subcutaneously at the right flank
with the Karpas422 tumor cells (5.times.10.sup.6) in 0.2 ml of PBS
with Matrigel (1:1) for tumor development. Day 23 after tumor
inoculation was as day 0 after the start of treatment when the
average tumor size reached approximately 300 mm.sup.3. Each group
consisted of 10 tumor-bearing mice.
[0172] Tumor Measurements
[0173] Tumor size was measured three times weekly in two dimensions
using a caliper, and the volume was expressed in mm.sup.3 using the
formula: V=0.536 a.times.b.sup.2 where a and b are the long and
short diameters of the tumor, respectively. The tumor size was then
used for calculations of T/C values. The T/C value (in percent) is
an indication of antitumor effectiveness; T and C are the mean
volumes of the treated and control groups, respectively, on a given
day. TGI was calculated for each group using the formula: TGI
(%)=[1-(Ti-T0)/(Vi-V0)].times.100; Ti is the average tumor volume
of a treatment group on a given day, T0 is the average tumor volume
of the treatment group on the day of treatment start, Vi is the
average tumor volume of the vehicle control group on the same day
with Ti, and V0 is the average tumor volume of the vehicle group on
the day of treatment start.
[0174] Experimental Endpoint and Sample Collection
[0175] 1). Plasma, tumor and muscle in EPZ-6438 group were
collected on day 16 after the start of treatment at 6 h after
dosing. Plasma, tumor and muscle in vehicle, CPI-524369, CPI-524416
and CPI-591780 groups were collected on day 25 after the start of
treatment at 1 h after dosing. 2). All the blood was taken from
each animal with EDTA-K2 as anticoagulant. Plasma was divided into
two parts. The first part was for PK analysis; the second part was
frozen for backup. 3). Tumor was divided into three parts. The
first part was snap-frozen for PK; the second part was snap-frozen
for PD analysis; the third part was frozen for backup. 4). Muscle
was divided into two parts. The first part was snap-frozen for PK;
the second part was frozen for backup.
[0176] Tumor Growth Inhibition Analysis
TABLE-US-00027 TABLE 4 Tumor growth inhibition calculation for
Compounds 362 and 365 in the karpass422 xenograft model calculated
based on tumor volume measurements on day 25 or day 16 after the
start of treatment Tumor Size (mm.sup.3).sup.a T/C.sup.b TGI
Treatment on day 25 (%) (%) Significance.sup.d Vehicle 1704 .+-.
123 -- -- -- Compound 362 385 .+-. 66 22.59 92.75 *** (160 mg/kg)
Compound 365 319 .+-. 67 18.72 97.25 *** (160 mg/kg) Note:
.sup.aMean .+-. SEM. .sup.bTumor Growth Inhibition is calculated by
dividing the group average tumor volume for the treated group by
the group average tumor volume for the control group (T/C). For a
test article to be considered to have anti-tumor activity, T/C must
be 0.5 or less. .sup.dStatistically significant difference (one-way
ANOVA), vs vehicle: *** p < 0.001.
* * * * *