Note: Descriptions are shown in the official language in which they were submitted.
CA 02721837 2015-07-30
DESCRIPTION
ANTIOXIDANT INFLAMMATION MODULATORS: OLEANOLIC ACID
DERIVATIVES WITH SATURATION IN THE C-RING
BACKGROUND OF THE INVENTION
1 0 I. Field of the Invention
The present invention relates generally to the fields of biology and medicine.
More
particularly, it concerns compounds and methods for the treatment and
prevention of diseases
such as those associated with oxidative stress and inflammation.
11. Description of Related Art
Many serious and intractable human diseases are associated with dysregulation
of
inflammatory processes, including diseases such as cancer, atherosclerosis,
and diabetes,
which were not traditionally viewed as inflammatory conditions. Similarly,
autoimmune
diseases such as rheumatoid arthritis, lupus, psoriasis, and multiple
sclerosis involve
inappropriate and chronic activation of inflammatory processes in affected
tissues, arising
from dysfunction of self vs. non-self recognition and response mechanisms in
the immune
system. In neurodegenerative diseases such as Alzheimer's and Parkinson's
diseases, neural
damage is correlated with activation of microglia and elevated levels of pro-
inflammatory
proteins such as inducible nitric oxide synthase (iNOS).
One aspect of inflammation is the production of inflammatory prostaglandins
such as
prostaglandin E, whose precursors are produced by the enzyme cyclo-oxygenasc
(COX-2).
High levels of COX-2 are found in inflamed tissues. Consequently, inhibition
of COX-2 is
known to reduce many symptoms of inflammation and a number of important anti-
inflammatory drugs (e.g., ibuprofen and celecoxib) act by inhibiting COX-2
activity. Recent
research, however, has demonstrated that a class of cyclopentenone
prostaglandins (e.g., 15-
deoxy prostaglandin J2, a.k.a. PGJ2) plays a role in stimulating the
orchestrated resolution of
inflammation. COX-2 is also associated with the production of
cyclopcntenone
1
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
prostaglandins. Consequently, inhibition of COX-2 may interfere with the full
resolution of
inflammation, potentially promoting the persistence of activated immune cells
in tissues and
leading to chronic, "smoldering" inflammation. This effect may be responsible
for the
increased incidence of cardiovascular disease in patients using selective COX-
2 inhibitors for
long periods of time. Corticosteroids, another important class of anti-
inflammatory drugs,
have many undesirable side effects and frequently are not suitable for chronic
use. Newer
protein-based drugs, such as anti-TNF monoclonal antibodies, have proven to be
effective for
the treatment of certain autoimmune diseases such as rheumatoid arthritis.
However, these
compounds must be administered by injection, are not effective in all
patients, and may have
severe side effects. In many severe forms of inflammation (e.g., sepsis, acute
pancreatitis),
existing drugs are ineffective. In addition, currently available drugs
typically do not have
significant antioxidant properties, and are not effective in reducing
oxidative stress associated
with excessive production of reactive oxygen species and related molecules
such as
peroxynitrite. Accordingly, there is a pressing need for improved therapeutics
with
antioxidant and anti-inflammatory properties.
A series of synthetic triterpenoid analogs of oleanolic acid have been shown
to be
inhibitors of cellular inflammatory processes, such as the induction by IFN-y
of inducible
nitric oxide synthase (iNOS) and of COX-2 in mouse macrophages. See Honda et
al.
(2000a); Honda et al. (2000b), and Honda et al. (2002), which are all
incorporated herein by
reference. For example, one of these, 2-cyano-3,12-dioxooleane-1,9(11)-dien-28-
oic acid
methyl ester (CDDO-Me), is currently in clinical trials for a variety of
disorders related to
inflammation, including cancer and diabetic nephropathy. The pharmacology of
these
molecules is complex, as they have been shown to affect the function of
multiple protein
targets and thereby modulate the function of several important cellular
signaling pathways
related to oxidative stress, cell cycle control, and inflammation (e.g.,
Dinkova-Kostova et al.,
Ahmad et al., 2006; Ahmad et al., 2008; Liby et al.,). Given that the
biological activity
profiles of the known oleanolic acid derivatives vary, and in view of the wide
variety of
diseases that may be treated with compounds having potent antioxidant and anti-
inflammatory effects, it is desirable to synthesize new candidates for the
treatment or
prevention of disease.
2
CA 02721837 2010-10-15
WO 2009/129545 PCT/US2009/041170
SUMMARY OF THE INVENTION
The present disclosure provides new compounds with antioxidant and anti-
inflammatory properties, methods for their manufacture, and methods for their
use.
Compounds covered by the generic or specific formulas below or specifically
named are
referred to as "compounds of the invention," "compounds of the present
disclosure," "the
present oleanolic acid derivatives" in the present application.
In some aspects, the disclosure provides compounds of the formula:
R10 R11
R9
X R8 20
21
% 19
%
12 % ,- - --Z 18
,
, %' 22
11
% ,' 7
/
R1 13% --
CH3 CH3 Y
1 9 14
R216_
.''' z
_
2r 10 8 = 15
' CH3
1
-- 3 ' 5 7
R3 '... -.......... .- -
4 Z 6
_
0
H '
R7
1
1
1
1
R4 R5 R6
(I),
wherein:
Y is cyano, heteroaryl(c<12), substituted heteroaryl(c<12), or ¨C(0)Ra,
further wherein
Ra is:
hydrogen, hydroxy, halo, amino, hydroxyamino, azido, silyl or
mercapto;
alkyl(c<12), alkenyl(c<12), alkynyl(c<12),
aryl(c12), aralkyl(c l2),
heteroaryl(c<12), heteroaralkyl(c<12), alkOXY(C<12), alkenyl-
oxy(ci2), alkynyloxy(ci2), aryloxy(c12),
aralkoxy(c 12),
heteroaryloxy(c<12)5 heteroaralkoxy(c<12),
acyloxy(c 12),
alkylamino(c<12)5 dialkylamino(C<12)5
alk0 XYaMin0(C 1 2),
alkenylamino(C<12)5 alkynylamino(c<12),
arylamino(c<12)5
aralkylamino(c<12)5 heteroarylamino(C<12)5
heteroaralkyl-
amino(C<12), alkylsulfonylamino(C<12), amido(C<12), alkyl-
3
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
thio(c<12), alkenylthio(c<12)5 alkynylthio(c<12), arylthio(c<12)5
aralkylthio(c<12), heteroarylthio(c<12), heteroaralkylthio(c<12),
acylthio(c<12), alkylammonium(c<12), alkylsulfonium(c<12)5
alkylsilyl(c<12), or a substituted version of any of these groups;
Or
Ra comprises a nitrogen atom that is also attached to carbon atom 13
and Rd to form:
H3c CH3
Rd 20
\ 19 21
0
18
22
11
13 1110 17
42) 16
¨ ¨ 15
CH3
wherein Rd is alkyl(c<8), alkenyl(c<8), aryl(cA, aralkyl(c<8),
heteroaryl(c<g), heteroaralkyl(c<8), or a substituted
version of any of these groups;
Z is a single or double bond, ¨0¨ or ¨NRe¨, wherein Re is hydrogen, hydroxy,
alkyl(c<8) or alkoxy();
X is ORb, NRbRe, or SRb, wherein Rb and Re are each independently:
hydrogen or hydroxy;
alkyl(c<8), aryl(c8), aralkyl(ca), acyl(cA), alkoxy(cA), aryloxy(8),
acyloxy(ca),
alkylamino(c<8), arylamino(c<8), amido(c<8), or a substituted version of
any of these groups; or
a substituent convertible in vivo to hydrogen;
provided that Rb is absent when the atom to which it is bound is part of a
double bond, further provided that when Rb is absent the atom to which
it is bound is part of a double bond;
R1 is:
hydrogen, cyano, hydroxy, halo or amino; or
alkyl(c<8), alkenyl(c<8), alkynyl(cA, aryl(c<8), aralkyl(cA, heteroaryl(cA,
heteroaralkyl(cA, acyl(c8), alkoxy(ca), aryloxy(ca), acyloxy(ca),
4
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
alkylamino(c<8), arylamino(c<8), amido(c<8), or a substituted version of
any of these groups;
R2 is:
cyano, hydroxy, halo or amino; or
fluoroalkyl(c<8), alkenyl(c<8), alkynyl(c<8), aryl(c<8), heteroaryl(c<8),
acYl(c<8),
alkoxy(c8), aryloxy(c<8), acyloxy(c<8), alkylamino(c<s), arylamino(c<8),
amido(c<8), or a substituted version of any of these groups;
R3 is:
absent or hydrogen;
alkyl(c<g), aryl(c<8), aralkyl(c<8), acyl(c<8), or a substituted version of
any of
these groups; or
a substituent convertible in vivo to hydrogen;
provided that R3 is absent when the oxygen atom to which it is bound is part
of
a double bond, further provided that when R3 is absent the oxygen
atom to which it is bound is part of a double bond;
R4 and R5 are each independently alkyl(c<8) or substituted alkyl(c<8);
R6 is hydrogen, hydroxy or oxo;
R7 is hydrogen or hydroxy; and
Rg, R95 Rio and R11 are each independently hydrogen, hydroxy, alkyl(c<8),
substituted
alkyl(c<8), alkoxy(c<8) or substituted alkoxrc<8);
or pharmaceutically acceptable salts, esters, hydrates, solvates, tautomers,
prodrugs, or optical
isomers thereof
5
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
In some embodiments, the compound is further defined as:
H3C CH3
1X 20
19 21
18
22
11 ,'"`.
,
R1 1
CH3 CH3
1 9 14
R2 16
0'
2
8 = 16
r
CH3
5 7
R3
" 4 6
0 R7
R4 R5 R6 (H),
wherein:
Y is cyano, or ¨C(0)Ra, further wherein:
5 Ra is:
hydrogen, hydroxy, halo, amino, hydroxyamino, azido or mercapto; or
alkenyl(c12), alkynyl(c12), aryl(c12), aralkyl(c12),
heteroaryl(c<12), heteroaralkyl(c<12), alkOXY(C<12), alkenyl-
OXY(C12), alkynYloXY(C12)5
arY1OXY(12), aralkoxy(c12) 5
1 0 heteroaryloxy(c<12)5 heteroaralkoxy(c<12)5
acyloxy(c12)5
alkylamino(c<12)5 dialkylamino(c<12)5
alkoxyamino(c<12),
alkenylamino(c<12)5 alkynylamino(c<12),
arylamino(c<12)5
aralkylamino(c<12)5 heteroarylamino(c<12)5
heteroaralkyl-
amino(c<12), alkylsulfonylamino(c<12), alllid0(C<12), alkyl-
thio(C<12), alkenylthio(c<12)5 alkynylthio(c<12) 5 arylthio(c< 12) 5
aralkylthio(c<12), heteroarylthio(c<12), heteroaralkylthio(c<12),
acylthio(c<12)5 alkYlaMMOniUM(C<12)5 alkylsulfonium(c<12)5
alkylsilyl(c<12), or a substituted version of any of these groups;
X is ORb, NRbRc, or SRb, wherein Rb and R, are each independently:
hydrogen or hydroxy;
6
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
alkyl(cA, aryl(c8), aralkyl(cA, acyl(cA), alkoxy(cs8), aryloxy(c8),
acyloxrc<8), alkylamino(c<s), arylamino(cA, amido(c<8), or a
substituted version of any of these groups; or
a substituent convertible in vivo to hydrogen;
provided that Rb is absent when the atom to which it is bound is part of
a double bond, further provided that when Rb is absent the atom
to which it is bound is part of a double bond;
R1 is:
hydrogen, cyano, hydroxy, halo or amino; or
alkyl(c<8), alkenyl(c<8), alkynyl(c<8), aryl(c<8), aralkyl(cA,
heteroaryl(c<8),
heteroaralkyl(8), acyl(c8), alkoxy(c8), aryloxy(c<8), acyloxy(c8),
alkylamino(c<8), arylamino(c<8), amido(c<8), or a substituted version of
any of these groups;
R2 is:
cyano, hydroxy, halo or amino; or
fluoroalkyl(cA, alkenyl(c<8), alkynYl(c<8), aryl(cA, heteroaryl(c<8),
acyl(c<8),
alkoxy(c<8), aryloxy(c_8), acyloxy(c<8), alkylamino(c<8), arylamino(c<s),
amido(c<8), or a substituted version of any of these groups;
R3 is:
absent or hydrogen;
alkyl(c<8), aryl(c<g), aralkyl(c<8), acyl(c<8), or a substituted version of
any of
these groups; or
a substituent convertible in vivo to hydrogen;
provided that R3 is absent when the oxygen atom to which it is bound is part
of
a double bond, further provided that when R3 is absent the oxygen
atom to which it is bound is part of a double bond;
R4 and R5 are each independently alkyl(c<8) or substituted alkyl(c<8); and
R6 and R7 are each independently hydrogen or hydroxy;
or pharmaceutically acceptable salts, esters, hydrates, solvates, tautomers,
prodrugs, or optical
isomers thereof
7
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
In some embodiments, the compound is further defined as:
H3C CH3
19 20 21
12 18
19
"
R1 11
CH 3 C H3
1 9
R2 16
2 10
i5
7 = 15
CH 3
0 H 6 8
R4 C H3 OM,
wherein:
Y is cyano or ¨C(0)Ra, further wherein:
Ra is:
hydrogen, hydroxy, halo, amino, azido, mercapto or silyl; or
alkyl(c<12), alkenyl(c<12), alkynYl(c<12), aryl(c12), aralkyl(c12),
heteroaryl(c<12), heteroaralkyl(c<12), alkOXY(C<12), alkenyl-
oxy(c12), alkyoyloxY(c12), aryloxy(12), aralkoxy(c12),
heteroaryloxy(c<12)5 heteroaralkoxy(c<12)5 acyloxy(c 12)5
alkylamino(c<12), alkoxyamino(c<12)5
dialkylamino(c<12),
alkenylamino(c<12)5 alkynylamino(c<12)5
arylamino(c<12)5
aralkylamino(c<12)5 heteroarylamino(c<12)5
heteroaralkyl-
amino(c<12), amido(c<12), or a substituted version of any of these
groups;
X is ORb, NRbRc, or SRb, wherein Rb and R, are each independently:
hydrogen or hydroxy;
alkyl(c8), aryl(c8), aralkyl("8), acyl(cA), alkoxy(cs8), aryloxy(c8),
acyloxrc<8), alkylamino(c<8), arylamino(cA), amido(c<8), or a
substituted version of any of these groups; or
a substituent convertible in vivo to hydrogen;
8
CA 02721837 2010-10-15
WO 2009/129545 PCT/US2009/041170
provided that Rb is absent when the atom to which it is bound is part of
a double bond, further provided that when Rb is absent the atom
to which it is bound is part of a double bond;
Ri is:
hydrogen, cyano, hydroxy, halo or amino; or
alkyl(cA, alkenyl(c<8), alkynyl(c8), aryl(c8), aralkyl(cs8), heteroaryl(cso,
heteroaralkyl(), acyl(c8), alkoxy(c_8), aryloxY(c8), acyloxy(c8),
alkylamino(c<8), arylamino(c<8), amido(c<8), or a substituted version of
any of these groups;
R2 is:
cyano, hydroxy, halo or amino; or
fluoroalkyl(c<8), alkenyl(c<8), alkynyl(c<8), aryl(c<8), heteroaryl(c<8),
acyl(c<8),
alkoxy(c8), aryloxy(c8), acyloxy(cA), alkylamino(cA, arylamino(C<8),
amido(c<8), or a substituted version of any of these groups; and
R4 is alkyl(c<8) or substituted alkyl(c<8);
or pharmaceutically acceptable salts, esters, hydrates, solvates, tautomers,
prodrugs, or optical
isomers thereof
In some embodiments, the compound is further defined as:
H3C CH3
X 20
19 21
1
12 18
oo 19
11
Ra
CH3 CH3
1 9 14
R2 16
2 10
õ = 15 0
CH3
7
0 H 6
H3C CH3 (IV),
wherein:
R4 is:
hydrogen, hydroxy, halo, or amino; or
9
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
alkyl(c<g), alkenyl(c<8), alkynyl(cA), ary1(8), aralkyl(cA), heteroary1(),
heteroaralkyl(c<8), alkoxy(c_8), alkenyloxy(cs8), alkynyloxy(c8),
aryloxy(c8), aralkoxy(8), heteroaryloxy(8), heteroaralkoxrco,
acyloxy(c,8), alkylamino(c<8), alkoxyamino(c<8), dialkylamino(c<8),
alkenylamino(c<8), alkynylamino(c<8), arylamino(c<8), aralkylamino(c<8),
heteroarylamino(c<s), heteroaralkylamino(c<8), amido(c<g), or a
substituted version of any of these groups;
X is ORb or NRbRc, wherein Rb and Rc are each independently:
hydrogen or hydroxy;
alkyl(c8), aryl(cA, aralkyl(c8), acyl(cA), alkoxrcA), aryloxy(c8),
acyloxy(8), alkylamino(c<8), arylamino(8), amido(cA, or a
substituted version of any of these groups; or
a substituent convertible in vivo to hydrogen;
provided that Rb is absent when the atom to which it is bound is part of
a double bond, further provided that when Rb is absent the atom
to which it is bound is part of a double bond; and
R2 is:
cyano, hydroxy, halo or amino; or
fluoroalkyl(c<8), alkenyl(c<8), alkYnYl(c<8), aryl(c<8), heteroary1(8),
acyl(c<8),
alkoxy(c8), aryloxy(c<8), acyloxy(c8), alkylamino(c<g), arylamino(cA),
amido(c<8), or a substituted version of any of these groups;
or pharmaceutically acceptable salts, esters, hydrates, solvates, tautomers,
prodrugs, or optical
isomers thereof
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
In some embodiments, the compound is further defined as:
H3C CH3
19 20 21
0
18
22
11 12 Ra
CH3 CH3
1 9
R2
2O10
6 16
7 CH3 0
0
H
8
H3C CH3 (V),
wherein:
Ra is:
5 hydrogen, hydroxy, halo, or amino; or
alkyl(c<g), alkenyl(c<8), alkYnYl(c<8), arY1(c<8), aralkyl(cA, heteroaryl(cA,
heteroaralkyl(c<8), alkoxy(c<8), alkenyloxy(c<8), alkynYloxy(c_8),
aryloxy(c<8), aralkoxy(c<8), heteroaryloxy(c<8), heteroaralkoxy(c<8),
acyloxy(c<8), alkylamino(c<8), alkoxyamino(c<8), dialkylamino(c<s),
10 alkenylamino(c<8), alkynylamino(c<8), arylamino(c<8),
aralkylamino(c<8),
heteroarylamino(c<8), heteroaralkylamino(c<8), amido(c<8), or a
substituted version of any of these groups; and
R2 is:
cyano or fluoro; or
15 fluoroalkyl(c<5), alkenyl(c<5), alkynyl(c<5), heteroary1(5),
acyl(c<5), acyloxy(c5),
amido(c<5), or a substituted version of any of these groups;
or pharmaceutically acceptable salts, esters, hydrates, solvates, tautomers,
prodrugs, or optical
isomers thereof
11
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
In some embodiments, the compound is further defined as:
0 40
Ra
NC Alp,*
0
0
(VI),
wherein Ra is:
hydrogen, hydroxy, halo or amino; or
alkyl(c<8), alkenyl(c<8), alkynyl(c<8), aryl(cA), aralkyl(8), heteroaryl(c8),
heteroaralkyl(c<g), alkoxy(c_8), alkenyloxy(c8), alkYnYloxY(8), aryloxy(c8),
aralkoxy(c<8), heteroaryloxy(c<8),
heteroaralkoxy(c<8), acyloxy(c8),
alkylamino(c<8), alkoxyamino(c<8), dialkylamino(c<8), alkenylamino(c<8),
alkynylamino(c<8), arylamino(c<8), aralkylamino(c<8), heteroarylamino(c<s),
heteroaralkylamino(c<8), amido(c<8), or a substituted version of any of these
groups;
or pharmaceutically acceptable salts, esters, hydrates, solvates, tautomers,
prodrugs, or optical
isomers thereof.
In some embodiments, the compound is further defined as:
ip
R a
NC 004P
0
0 H-
(VII),
wherein Ra is alkoxy(c 1 -4), alkylamino(ci_4), alkoxyamino(ci_4),
dialkylamino(c2_4), or a
substituted version of any of these groups; or pharmaceutically acceptable
salts, esters,
hydrates, solvates, tautomers, prodrugs, or optical isomers thereof
12
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
In some embodiments, the compound is further defined as:
i
=
O
R a
NC .0410iW
0
0 =,,,,H-
(VIM,
wherein Ra is alkyl(ci_4) or aralkoxy(c7_8) or a substituted version of either
of these groups; or
pharmaceutically acceptable salts, esters, hydrates, solvates, tautomers,
prodrugs, or optical
isomers thereof
In some embodiments, the compound is further defined as:
O
R a
N C .0410110
0
: (IX),
wherein Ra is hydrogen, hydroxy, amino, dimethylamino, methyl, methoxy,
methoxyamino,
benzyloxy, or 2,2,2-trifluoroethylamino; or pharmaceutically acceptable salts,
hydrates,
solvates, tautomers, or optical isomers thereof
In some embodiments, the compound is further defined as:
..
=
0=O R a
NC so ,
0
(X),
wherein Ra is hydrogen, hydroxy, amino, methoxy, or 2,2,2-trifluoroethylamino;
or
pharmaceutically acceptable salts, hydrates, solvates, tautomers, or optical
isomers thereof
13
CA 02721837 2010-10-15
WO 2009/129545 PCT/US2009/041170
In some embodiments, the compound is further defined as:
i
Rd
0 \N 0
NC
WA"dIP.
0 ,H
(XI),
wherein Rd is alkyl(c<g), alkellyl(c<g), aryl(c<g), aralkyl(c<8),
heteroaryl(c<8), heteroaralkyl(c<8), or
a substituted version of any of these groups; or pharmaceutically acceptable
salts, hydrates,
solvates, tautomers, or optical isomers thereof.
In some embodiments, the compound is further defined as:
i
0 O
NC leo
0,0 Y
: (XII),
wherein Y is heteroaryl(c<8) or a substituted heteroaryl(c<8); or a
pharmaceutically acceptable
salts, hydrates, solvates, tautomers, or optical isomers thereof
In some embodiments, the compound is further defined as:
H3C CH3
19 20 21
0
18
11 12 22
CH3 CH3 Y
1 9
R2 EIO 16
2 10 8 15
CH3
.' 5
R3... ....... _ 7
=
0
H 6
R4 CH3 (XIII),
wherein:
wherein Y is cyano or ¨C(0)Ra, further wherein:
14
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
Ra is:
hydrogen, hydroxy, halo, amino, azido, mercapto or silyl; or
alkyl(c<12), alkenyl(c<12), alkynyl(c<12)5
aryl(c12), aralkyl(c 12),
heteroaryl(c<12), heteroaralkyl(c<12), alkOXY(C<12), alkenyl-
oxrc12), alkynYloxY(12), aryloxrc12), aralkoxrc12),
heteroaryloxrc<12)5 heteroaralkoxrc<12)5
acyloxrc12)5
alkylamino(c< 12), alkoxyamino(c<12)5
dialkylamino(c<12),
alkenylamino(c<12)5 alkynylamino(c<12)5
arylamino(c< 12),
aralkylamino(C<12)5 heteroarylamino(C< 12),
heteroaralkyl-
amino(c<12), amido(c<12), or a substituted version of any of these
groups;
R2 is:
cyano, hydroxy, halo or amino; or
fluoroalkyl(c<8), alkenyl(cA, alkynyl(c<8), aryl(c<8), heteroaryl(cA),
acyl(cA),
alkoxrc8), aryloxrc8), acyloxrc8), alkylamino(cA, arylamino(cA,
amido(c<8), or a substituted version of any of these groups;
R3 is:
absent or hydrogen;
alkyl(c<g), aryl(c<8), aralkyl(c<8), acyl(c<8), or a substituted version of
any of
these groups; or
a substituent convertible in vivo to hydrogen;
provided that R3 is absent when the oxygen atom to which it is bound is part
of
a double bond, further provided that when R3 is absent the oxygen
atom to which it is bound is part of a double bond; and
R4 is alkyl(c<8) or substituted alkyl(c<8);
or pharmaceutically acceptable salts, esters, hydrates, solvates, tautomers,
prodrugs, or optical
isomers thereof.
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
In some embodiments, the compound is further defined as:
=
Alile
Y
R2
R3
0 H
(XIV),
wherein:
Y is cyano or ¨C(0)Ra, wherein
Ra is:
hydrogen, hydroxy, halo, amino, hydroxyamino, azido or mercapto; or
alkyl(c<12), alkenyl(c<12), alkynyl(c<12), aryl(c<12), aralkyl(c<12),
heteroaryl(c<12), heteroaralkyl(c<12), alkOXY(C<12), alkenyl-
OXy(c12), alkynyloxrc<12), aryloxrc<12), aralkoxrc<12),
heteroaryloxrc<12)5 heteroaralkoxrc<12)5 acyloxrc< 12)5
alkylamino(c<12), alkoxyamino(c<12)5
dialkylamino(c<12),
alkenylamino(c< 12)5 alkynylamino(c<12)5
arylamino(c< 12)5
aralkylamino(c<12)5 heteroarylamino(c<12)5
heteroaralkyl-
amino(c<12), alkylsulfonylamino(c<12), amido(c<12), or a
substituted version of any of these groups;
R2 is:
cyano, hydroxy, halo or amino; or
fluoroalkyl(c<8), alkenyl(c<8), alkynyl(cA, aryl(cA, heteroaryl(cA),
acyl(c<8),
alkoxy(c8), aryloxrc8), acyloxrc8), alkylamino(cA, arylamino(c<8),
amido(c<8), or a substituted version of any of these groups; and
R3 is:
hydrogen;
alkyl(c<8), aryl(c<8), aralkyl(c<8), acyl(c<8), or a substituted version of
any of
these groups; or
a substituent convertible in vivo to hydrogen;
or pharmaceutically acceptable salts, esters, hydrates, solvates, tautomers,
prodrugs, or optical
isomers thereof
16
CA 02721837 2010-10-15
WO 2009/129545 PCT/US2009/041170
In some embodiments, the compound is further defined as:
H3C CH3
1X 20
19 21
1
1
12 18
19
,'"`,, 7
R1 11
CH3 CH3 C
1 9
R2 16 N
:
=
2 0 10 6 8 = 15
CH3
_ 7
0 =
H
R4 CH3 (XV),
wherein:
X is ORb, NRbRc, or SRb, wherein Rb and Rc are each independently:
5 hydrogen;
alkyl(c<8), aryl(c<8), aralkyl(c<s), acyl(c<8), or a substituted version of
any
of these groups; or
a substituent convertible in vivo to hydrogen;
provided that Rb is absent when the atom to which it is bound is part of
a double bond, further provided that when Rb is absent the atom
to which it is bound is part of a double bond;
R1 is:
hydrogen, cyano, hydroxy, halo or amino; or
alkyl(cA, alkenyl(cA, alkynyl(c8), aryl(c8), aralkyl(cA, heteroaryl(c<8),
heteroaralkyl(c<8), acyl(c8), alkoxy(c_8), aryloxy(c8), acyloxy(c8),
alkylamino(c<8), arylamino(c<8), amido(c<8), or a substituted version of
any of these groups;
R2 is:
cyano, hydroxy, halo or amino; or
fluoroalkyl(cA, alkenyl(cA, alkYnYl(c<8), aryl(c<8), heteroaryl(c<8),
acyl(c<8),
alkoxy(c8), aryloxy(c8), acyloxy(c8), alkylamino(cA, arylamino(cA,
amido(c<8), or a substituted version of any of these groups; and
17
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
R4 is alkyl(c<8) or substituted alkyl(c<8);
or pharmaceutically acceptable salts, esters, hydrates, solvates, tautomers,
prodrugs, or optical
isomers thereof
In some embodiments, the compound is further defined as:
H3C CH3
19 20 21
13 18
22
11 17
CH3 CH3
1 9
R2 16
2 1 0 6 8 E 15
CH3
7
0
H
5 H3C CH3 (XVI),
wherein:
X is ORb or NRbRc, wherein Rb and R, are each independently:
hydrogen;
alkyl(c<8), aryl(c<8), aralkyl(c<8), acyl(c<8), or a substituted version of
any
of these groups; or
a substituent convertible in vivo to hydrogen;
provided that Rb is absent when the atom to which it is bound is part of
a double bond, further provided that when Rb is absent the atom
to which it is bound is part of a double bond; and
R2 is:
cyano, hydroxy, halo or amino; or
fluoroalkyl(cA, alkenyl(cA, alkYnYl(c<8), aryl(c<8), heteroaryl(cA), acyl(c8),
alkoxy(c8), aryloxy(c8), acyloxy(8), alkylamino(c<8), arylamino(cA,
amido(c<8), or a substituted version of any of these groups;
or pharmaceutically acceptable salts, esters, hydrates, solvates, tautomers,
prodrugs, or optical
isomers thereof
18
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
In some embodiments, the compound is further defined as:
H3C CH3
19 20 21
OH
18
11 12 22
Ra
CH3 CH3
1 9
R2
2 10 6 16 8 15 0
CH3
15 7
0 H
H3C CH3
wherein:
Ra is:
hydrogen, hydroxy, halo, amino, azido, mercapto or silyl; or
alkyl(c<12), alkenyl(12), alkynyl(c12), ary1(12), aralkyl(c12),
heteroaryl(c<12),
heteroaralkyl(c<12), alkOXY(C<12), alkelly1OXY(C<12), alkynyloxrc<12),
aryloxy(c<12), aralkoxy(c<12), heteroaryloxrc<12), heteroaralkoxrc<12),
alkylamino(c<12),
acyloxrc<12)5
alkoxyamino(c<12), dialkylamino(c< 1 2)5
1 0 alkenylamino(c<12)5 alkynylamino(c<12)5
arylamino(c< 12)5
aralkylamino(c<12) 5 heteroarylamino(c<12)5 heteroaralkylamino(c< 1 2)5
amido(c<12), or a substituted version of any of these groups;
R1 is:
hydrogen, cyano, hydroxy, halo or amino; or
1 5 alkyl(c<8), alkenyl(c<g), alkyllyl(cA), aryl(c<8), aralkyl(c<8)
heteroaryl(c<8),
heteroaralkyl(c<8), acyl(c<8), alkoxy(c8), aryloxy(c<8), acyloxy(8),
alkylamino(C<g), arylamino(c<8), amido(c<8), or a substituted version of
any of these groups;
R2 is:
20 cyano, hydroxy, halo or amino; or
19
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
fluoroalkyl(c<8), alkenyl(c<8), alkYnYl(cA), aryl(cs8), heteroaryl(cA),
acyl(cso,
alkoxy(cm, aryloxrca), acyloxrc<8), alkylamino(cA, arylamino(c<o,
amido(c<8), or a substituted version of any of these groups; and
or pharmaceutically acceptable salts, esters, hydrates, solvates, tautomers,
prodrugs, or optical
isomers thereof.
In some embodiments, the compound is further defined as:
H OH el
R a
NC leo .z
0
0
(XVIII),
wherein Ra is:
hydrogen, hydroxy, halo or amino; or
alkyl(6), aryl(c<8), aralkyl(c<8), heteroaryl(cA, alkoxy(6), aryloxrcg),
aralkoxrcso,
alkylamino(c<0, alkoxyamino(c<6), alkoxyamino(c<6), dialkylamino(c<6),
arylamino(c<8), aralkylamino(c<s), heteroarylamino(C<6), heteroarylamino(cso,
amido(c<6), or a substituted version of any of these groups;
or pharmaceutically acceptable salts, esters, hydrates, solvates, tautomers,
prodrugs, or optical
isomers thereof
In some aspects, the disclosure provides compounds of the formula:
H3C CH3
19 20 21
12 18
19
11 ,.".
R1
CH3 CH3
1 9 14
N40_
5 6 8 = 15
CH3
16
7
0 3 -
H
H3C CH3 (XIX),
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
wherein:
Y is cyano or -C(0)Ra, wherein
Ra is:
hydrogen, hydroxy, halo, amino, hydroxyamino, azido or mercapto; or
alkyl(c12), alkenyl(c12), alkynyl(c12), aryl(c12), aralkyl(c12),
heteroaryl(c<12), heteroaralkyl(c<12), alkOXY(C<12), alkenyl-
oxy(c12)5 alkynYloxY(c12)5 aryloxy(c12), aralkoxy(c12)5
heteroaryloxy(c<12)5
heteroaralkoxy(c<12)5 acyloxy(c<12)5
alkYlanainO(C<12), dialkylamino(c<12), alkoxyamino(c<12), alkenyl-
amino(c<12)5 alkynylamino(c<12)5 arylamino(c<12)5
aralkylamino(c<12)5
heteroarylamino(c<12)5 heteroaralkyl-
amino(c<12), alkylsulfonylamino(c<12), amido(c<12), alkyl-
thio(c<12), alkenylthio(c<12), alkynylthio(c<12), arylthio(c<12),
aralkylthio(c<12), heteroarylthio(c<12), heteroaralkylthio(c<12),
acylthio(c<12), alkylammonium(c<12), alkylsulfonium(c<12)5
alkylsilyl(c<12), or a substituted version of any of these groups;
X is ORb, NRbRc, or SRb, wherein Rb and R, are each independently:
hydrogen;
alkyl(c<8), aryl(c<8), aralkyl(c<8), acyl(c<8), or a substituted version of
any
of these groups; or
provided that Rb is absent when the atom to which it is bound is part of
a double bond, further provided that when Rb is absent the atom
to which it is bound is part of a double bond; and
R1 is:
hydrogen, cyano, hydroxy, halo or amino; or
alkyl(c<8), alkenyl(c<8), alkynyl(c<8), aryl(c<8), aralkyl(cs8),
heteroaryl(c<8),
heteroaralkyl(cA, acyl(c8), alkoxy(ca), aryloxY(c8), acyloxy(c8),
alkylamino(c<s), arylamino(c<8), amido(c<8), or a substituted version of
any of these groups;
or salts, esters, hydrates, solvates, tautomers, or optical isomers thereof
21
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
In some aspects, the disclosure provides compounds of the formula:
H3C CH3
X 20
19 21
13 18
19
11 17
R1
CH3 CH3
1 9 14
16
= 10 8 E 15
CH3
7
0 H 6
H3C CH3 POO,
wherein:
Y is cyano or -C(0)Ra, wherein
Ra is:
hydrogen, hydroxy, halo, amino, hydroxyamino, azido or mercapto; or
alkenyl(c12), alkynyl(c12), aryl(c12), aralkyl(c12),
heteroaryl(c<12), heteroaralkyl(c<12), alkOXY(C<12), alkenyl-
OXy(c12), alkynyloxY(c12), aryloxy(c12), aralkoxy(c12),
heteroaryloxy(c<12)5 heteroaralkoxy(c<12), acyloxy(c<12)5
alkylamino(c<12), dialkylamino(c<12)5
alkoxyamino(c<12),
alkenylamino(c<12) 5 alkynylamino(c<12) 5
arylamino(c<12) 5
aralkylamino(c<12)5 heteroarylamino(c<12)5
heteroaralkyl-
amino(c<12), alkylsulfonylamino(c<12), anlid0(c<12), alkyl-
thio(c<12), alkenylthio(c<12), alkynylthio(c<12), arylthio(c<12),
aralkylthio(c<12), heteroarylthio(c<12), heteroaralkylthio(c<12)5
aCYlthiO(C 12), alkylammonium(c<12),
alkylsulfonium(c<12),
alkylsilyl(c<12), or a substituted version of any of these groups;
X is ORb, NRbRc, or SRb, wherein Rb and R, are each independently:
hydrogen;
alkyl(c<8), aryl(c<8), aralkyl(c<s), acyl(c<8), or a substituted version of
any
of these groups; or
22
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
provided that Rb is absent when the atom to which it is bound is part of
a double bond, further provided that when Rb is absent the atom
to which it is bound is part of a double bond; and
R1 is:
hydrogen, cyano, hydroxy, halo or amino; or
alkyl(c<8), alkenyl(c<8), alkynyl(cA), aryl(c8), aralkyl(cA), heteroaryl(cA),
heteroaralkyl(cA, acyl(c8), alkoxy(c_8), aryloxy(cs8), acyloxy(c8),
alkylamino(c<8), arylamino(c<8), amido(c<8), or a substituted version of
any of these groups;
R' is hydroxy, alkoxy(c<12), substituted alkoxy(c<12), aryloxy(c<12),
substituted
aryloxy(c<12), aralkoxy(c<12), substituted aralkoxy(c<12), aCylOXY(C<12), or
substituted acyloxy(c<12);
or salts, esters, hydrates, solvates, tautomers, or optical isomers thereof.
In a variation of each of the above embodiments containing a Z group, Z can be
a
single bond, ¨0¨, or ¨NH¨. In a variation of each of the above embodiments
containing an
X group, X can be ORb. In some variations, Rb is absent. In other variations,
Rb is hydrogen.
In other variations, X can be NRb. In some variations, Rb can be hydroxy. In a
variation of
each of the above embodiments containing a Y group, Y can be cyano or ¨C(0)Ra.
In some
variations, Ra can be hydroxy. In some variations, Ra can be alkoxy(c<6),
aryloxy(c<8),
aralkyloxy(c<8), or a substituted version of any of these groups. In some of
these variations,
Ra can be alkoxy(c2_6). In some of these variations, Ra can be alkoxy(c1_5) or
substituted
alkoxy(c1_5). In some of these variations, Ra can be alkoxy(c2_4) or
substituted alkoxv
(c2-4). In
some of these variations, Ra can be alkoxy(c1_4) or substituted alkoxy(c1_4).
In some of these
variations, Ra can be alkoxy(C1-2) or substituted alkoxy(c1_2). For example,
Ra can be methoxy.
In some variations, Ra can be amino. In some variations, Ra can be
alkylamino(ci-6),
alkoxyamino(c1_6), arylamino(c1-8), aralkylamino(c1_8), dialkylamino(c2_8), or
a substituted
version of any of these groups. In some of these variations, Ra can be
alkylamino(c2_6) or
substituted alkylamino(c2_6). In some of these variations, Ra can be
alkylamino(c3_6). In some
variations, Ra can be alkylamino(c1_5), dialkylamino(c2_6), or substituted
version of either of
these groups. In some variations, Ra can be alkylamino(c2_4),
dialkylamino(c2_5), or substituted
version of either of these groups. In some variations, Ra can be alkylamino(c1-
4) or substituted
alkylamino(c1_4). In some variations, Ra can be alkylamino(c1_3). In some of
these variations,
23
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
Ra can be methylamino or ethylamino. In some of these variations, Ra can be
substituted
alkylamino(c1_3). For example, Ra can be 2,2,2-trifluoroethylamino. In some of
these
variations, Ra can be alkyl(c1_5), aryl(c<g), aralkyl(c<8),
heteroaralkyl(c<8), or a substituted
version of any of these groups. In some of these variations, Ra can be
heteroaryl(ci_8) or
substituted heteroaryl(ci_8). For example, wherein Ra can be imidazolyl. In
some of these
variations, Ra can be ¨H.
In a variation of each of the above embodiments containing an R1 group, R1 can
be
¨H, ¨OH or ¨F. For example, R1 can be ¨H. In a variation of each of the above
embodiments containing an R2 group, R2 can be ¨CN. In some variations, R2 can
be a
substituted acyl(ci_3), such as ¨C(=0)NHS(=0)2CH3.
In some variations, R2 is
fluoroalkyl(c<8). For example, R2 can be ¨CF3. In other variations, R2 is not
fluoroalkyl(c<8).
In a variation of each of the above embodiments containing an R3 group, R3 can
be
hydrogen or acetyl. In another variation, R3 can be absent. In a variation of
each of the
above embodiments containing an R4 group, R4 can be methyl or hydroxymethyl.
In a
variation of each of the above embodiments containing an R6, R7, R8, or R9
group, R6, R75 R85
or R9 can independently be hydrogen. In a variation of each of the above
embodiments
containing an R10 or R11 group, R10 or R11 can independently be methyl. In a
variation of each
of the above embodiments containing an R' group, R' can be acetyloxy or
hydroxy.
In a variation of each of the above embodiments containing an Rd group, Rd can
be
alkyl(c1_5), aryl(c<8), aralkyl(c<8), heteroaralkyl(c<8), or a substituted
version of any of these
groups. In some variations, Rd can be alkyl(ci_4) or a substituted version
thereof. In some
variations, Rd can be alkyl(ci_3) or a substituted version thereof. In some
variations, Rd can be
alkyl(ci_2) or a substituted version thereof
Non-limiting examples of compounds provided by this invention include the
compounds according to the formulas shown below, as well as or
pharmaceutically
acceptable salts thereof In certain embodiments, these compounds are
substantially free
from other optical isomers thereof
24
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
.-
= O = O
00 0.,,Li
..,n3 040 0.CH3
NC es i
0 NC O. i
0
HO =,)---1
63101 63250
(402-02) (dh402) (402-46)
.s
O O o O
00 OH 00 CN
NC so i
0 NC es i
63196 63252
(402-51) (dh401) (402-57)
:
..
o O .ó
NH2 H
NC e00s i
0 NC 00glii i
0
63205 63206
(402-59) (402-64)
H,. OH O H0_, H O
0
0 0,
=00 0.0 H3
NC *0 i0 CH3 NC gek i
0
63207 63219
(402-66) (402-78)
CA 02721837 2010-10-15
WO 2009/129545 PCT/US2009/041170
i
0
=H,. OH O O0 H
N CF 3
......,..-CF3 NC . NC .00110
0 0
0 %)---1 ---
63102
63268
(404-02) (dh404)
.. i
= O = 0
H H
00 N'N '*.13 CH3 00 NCF3
NC *Iv
0 H NC O. _
0
n
--,
63264 63227
= O = O
I I
NC e joi= _ ,NH ,
N-N NC .4100
Nzze-CH3
63229 63230
.. F3cm
0 $N
,,,N,
N
NC o
e sop ___,I
A NC
SO
O.
63267 63237
.. i
NCNC 0 00 s_ iiN :0 4 . CH3
---\ so 10 MO 0
63274 63308
26
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
0 0
CH3
Adr 0 0 'CH3
NC 4041=Nri NC
0
0
0 0 7-7
63323 63325
HO,
0
NC
NØ NC
CH3 ilia 0,
CH3
0 akdkii
0
0 7-710 0 rr
63326 63295
0 0
NH 0
!III -cH3 = 0,CH3
NC es
0 NC
o IMO 0
0
63296 63263
HO
0
(1..CH3
NC es0
0
63289
Examples of specific compounds provided by the present disclosure include:
(4aS,6aR,6bR,8aR,12aR,14aR,14bS)-methyl 11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-
10,14-dioxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,12a,12b,13,14,14a,14b-
icosahydropicene-4a-
carboxylate,
(4aS,6aR,6bR,8aR,12aR,14aR,14bS)-methyl 11-cyano-10-hydroxy-2,2,6a,6b,9,9,12a-
heptamethy1-14-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,12,12a,12b,13,14,14a,14b-
icosahydropicene-4a-carboxylate,
27
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
(4aS,6aR,6bR,8aR,12aR,14aR,14bS)-11-cyano-2,2,6a,6b,9,9,12a-heptamethy1-10,14-
dioxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,12a,12b,13,14,14a,14b-icosahydropicene-
4a-
carboxylic acid,
(4aR,6aR,6bR,8a5,12a5,12bR,14bR)-4,4,6a,6b,11,11,14b-heptamethy1-3,13-dioxo-
3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14,14a,14b-icosahydropicene-2,8a-
dicarbonitrile,
(4a5,6aR,6bR,8aR,12aR,14aR,14bS)-11-cyano-2,2,6a,6b,9,9,12a-heptamethy1-10,14-
dioxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,12a,12b,13,14,14a,14b-icosahydropicene-
4a-
carboxamide,
(4aR,6aR,6bR,8a5,12a5,12bR,14bR)-8a-formy1-4,4,6a,6b,11,11,14b-heptamethy1-
3,13-dioxo-3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14,14a,14b-
icosahydropicene-2-
carbonitrile,
(4a5,6aR,6bR,8aR,12aR,14R,14aR,14bS)-methyl 11-cyano-14-hydroxy-
2,2,6a,6b,9,9,12a-heptamethy1-10-oxo-
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,12a,12b,13,14,14a,14b-icosahydropicene-4a-
carboxylate,
(4a5,6aR,6bR,8aR,12aR,14aR,14bS)-11-cyano-2,2,6a,6b,9,9,12a-heptamethy1-10,14-
dioxo-N-(2,2,2-trifluoroethyl)-
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,12a,12b,13,14,14a,14b-
icosahydropicene-4a-carboxamide,
(4a5,6aR,6bR,8aR,12aR,12bR,14R,14aR,14bS)-2,2,2-trifluoroethyl 11-cyano-14-
hydroxy-2,2,6a,6b,9,9,12a-heptamethy1-10-oxo-
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,12a,12b,13,14,14a,14b-icosahydropicene-4a-
carboxylate,
(4a5,6aR,6bR,8aR,12aR,12bR,14aR,14bS)-11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-
10,14-dioxo-N-(2,2,2-trifluoroethyl)-
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,12a,12b,13,14,14a,14b-
icosahydropicene-4a-carboxamide,
(4a5,6aR,6bR,8aR,12aR,12bR,14aR,14bS)-N'-acety1-11-cyano-2,2,6a,6b,9,9,12a-
heptamethy1-10,14-dioxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,12a,12b,13,14,14a,14b-
icosahydropicene-4a-carbohydrazide,
(4a5,6aR,6bR,8aR,12aR,12bR,14aR,14bS)-11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-
10,14-dioxo-N-(2,2,2-trifluoroethyl)docosahydropicene-4a-carboxamide,
(4aR,6aR,6bR,8aS,12aS,12bR,14aR,14bR)-4,4,6a,6b,11,11,14b-heptamethy1-3,13-
dioxo-8a-(2H-tetrazol-5-y1)-
3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14,14a,14b-
icosahydropicene-2-carbonitrile,
28
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
(4aR,6aR,6bR,8aS,12aS,12bR,14aR,14bR)-4,4,6a,6b,11,11,14b-heptamethy1-8a-(2-
methy1-2H-tetrazol-5-y1)-3,13-dioxo-
3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14,14a,14b-icosahydropicene-2-
carbonitrile,
(4aR,6aR,6bR,8a5,12a5,12bR,14aR,14bR)-4,4,6a,6b,11,11,14b-heptamethy1-8a-(5 -
methy1-1,3,4-oxadiazol-2-y1)-3,13-dioxo-
3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14,14a,14b-icosahydropicene-2-
carbonitrile,
and
(4aR,6aR,6bR,8a5,12a5,12bR,14aR,14bR)-4,4,6a,6b,11,11,14b-heptamethy1-8a-(5 -
methy1-1,3,4-thiadiazol-2-y1)-3,13-dioxo-
3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14,14a,14b-icosahydropicene-2-
carbonitrile,
(4aR,6aR,6bR,8a5,12a5,12bR,14aR,14bR)-8a-acety1-4,4,6a,6b,11,11,14b-
heptamethy1-
3,13-dioxo-3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14,14a,14b-
icosahydropicene-2-
carbonitrile,
(4a5,6aR,6bR,8aR,12aR,12bR,14aR,14bS)-benzyl 11-cyano-2,2,6a,6b,9,9,12a-
heptamethy1-10,14-dioxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,12a,12b,13,14,14a,14b-
icosahydropicene-4a-carboxylate,
(4a5,6aR,6bR,8aR,12aR,12bR,14aR,14bS)-11-cyano-N,N,2,2,6a,6b,9,9,12a-
nonamethy1-10,14-dioxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,12a,12b,13,14,14a,14b-
icosahydropicene-4a-carboxamide,
(4aS,6aR,6bR,8aR,12aR,12bR,14aR,14bS)-11-cyano-N-methoxy-2,2,6a,6b,9,9,12a-
heptamethy1-10,14-dioxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,12a,12b,13,14,14a,14b-
icosahydropicene-4a-carboxamide
(4a5,6aR,6bR,8aR,12aR,12bR,14aR,14bS)-methyl 11-cyano-14-(hydroxyimino)-
2,2,6a,6b,9,9,12a-heptamethy1-10-oxo-
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,12a,12b,13,14,14a,14b-icosahydropicene-4a-
carboxylate,
(4aR,6aR,6b5,8a5,12aR,15aR,15bR)-methyl 2-cyano-14-hydroxy-4,4,6a,6b,11,11,15b-
heptamethy1-3-oxo-3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,14,15,15a,15b-
icosahydrodinaphtho[1,2-b:2',1'-c/]oxepine-8a-carboxylate, and
(4aR,6aR,6b5,8a5,12aR,12bR,15aR,15bR)-methyl 2-cyano-4,4,6a,6b,11,11,15b-
heptamethy1-3,14-dioxo-
4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14,15,15a,15b-
icosahydro-3H-dinaphtho[1,2-b:2',1'-d]azepine-8a-carboxylate.
In some embodiments, compounds of the present disclosure are in the form of
pharmaceutically acceptable salts. In other embodiments, compounds of the
present
29
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
disclosure are not in the form of a pharmaceutically acceptable salts. In some
embodiments,
compounds of the present disclosure are in the form of a hydrate. In other
embodiments,
compounds of the present disclosure are not in the form of a hydrate. In some
embodiments,
compounds of the present disclosure are in the form of a solvate. In other
embodiments,
compounds of the present disclosure are not in the form of a solvate.
In some embodiments, compounds of the present disclosure can be esters of the
above
formulas. The ester may, for example, result from a condensation reaction
between a
hydroxy group of the formula and the carboxylic acid group of biotin. In other
embodiments,
compounds of the present disclosure are not an ester.
In some embodiments, the compounds of the present disclosure can be present as
a
mixture of stereoisomers. In other embodiments, the compounds of the present
disclosure are
present as single stereoisomers.
In some embodiments, compounds of the present disclosure may be inhibitors of
IFN-
y-induced nitrous oxide (NO) production in macrophages, for example, having an
IC50 value
of less than 0.2 M.
Other general aspects of the present disclosure contemplate a pharmaceutical
composition comprising as an active ingredient a compound of the present
disclosure and a
pharmaceutically acceptable carrier. The composition may, for example, be
adapted for
administration by a route selected from the group consisting of orally,
intraadiposally,
intraarterially, intraarticularly, intracranially, intradermally,
intralesionally, intramuscularly,
intranasally, intraocularally, intrapericardially,
intraperitoneally, intrapleurally,
intraprostaticaly, intrarectally, intrathecally, intratracheally,
intratumorally, intraumbilically,
intravaginally, intravenously, intravesicularlly, intravitreally, liposomally,
locally, mucosally,
orally, parenterally, rectally, subconjunctival, subcutaneously, sublingually,
topically,
transbuccally, transdermally, vaginally, in crèmes, in lipid compositions, via
a catheter, via a
lavage, via continuous infusion, via infusion, via inhalation, via injection,
via local delivery,
via localized perfusion, bathing target cells directly, or any combination
thereof In particular
embodiments, the composition may be formulated for oral delivery. In
particular
embodiments, the composition is formulated as a hard or soft capsule, a
tablet, a syrup, a
suspension, a wafer, or an elixir. In certain embodiments, the soft capsule is
a gelatin
capsule. Certain compositions may comprise a protective coating, such as those
compositions
formulated for oral delivery. Certain compositions further comprise an agent
that delays
absorption, such as those compositions formulated for oral delivery. Certain
compositions
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
may further comprise an agent that enhances solubility or dispersibility, such
as those
compositions formulated for oral delivery. Certain compositions may comprise a
compound
of the present disclosure, wherein the compound is dispersed in a liposome, an
oil in water
emulsion or a water in oil emulsion.
Yet another general aspect of the present disclosure contemplates a
therapeutic
method comprising administering a pharmaceutically effective compound of the
present
disclosure to a subject. The subject may, for example, be a human. These or
any other
methods of the present disclosure may further comprise identifying a subject
in need of
treatment.
Another method of the present disclosure contemplates a method of treating
cancer in
a subject, comprising administering to the subject a pharmaceutically
effective amount of a
compound of the present disclosure. The cancer may be any type of cancer, such
as a
carcinoma, sarcoma, lymphoma, leukemia, melanoma, mesothelioma, multiple
myeloma, or
seminoma. Other types of cancers include cancer of the bladder, blood, bone,
brain, breast,
central nervous system, colon, endometrium, esophagus, genitourinary tract,
head, larynx,
liver, lung, neck, ovary, pancreas, prostate, spleen, small intestine, large
intestine, stomach, or
testicle. In these or any other methods, the subject may be a primate. In
these or any other
methods, the subject may be a human. This or any other method may further
comprise
identifying a subject in need of treatment. The subject may have a family or
patient history of
cancer. In certain embodiments, the subject has symptoms of cancer. The
compounds of the
invention may be administered via any method described herein, such as
locally. In certain
embodiments, the compound is administered by direct intratumoral injection or
by injection
into tumor vasculature. In certain embodiments, the compounds may be
administered
systemically.
The compounds may be administered intravenously, infra-arterially,
intramuscularly, intraperitoneally, subcutaneously or orally, in certain
embodiments.
In certain embodiments regarding methods of treating cancer in a subject,
comprising
administering to the subject a pharmaceutically effective amount of a compound
of the
present disclosure, the pharmaceutically effective amount is 0.1 ¨ 1000 mg/kg.
In certain
embodiments, the pharmaceutically effective amount is administered in a single
dose per day.
In certain embodiments, the pharmaceutically effective amount is administered
in two or
more doses per day. The compound may be administered by contacting a tumor
cell during
ex vivo purging, for example. The method of treatment may comprise any one or
more of the
following: a) inducing cytotoxicity in a tumor cell; b) killing a tumor cell;
c) inducing
31
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
apoptosis in a tumor cell; d) inducing differentiation in a tumor cell; or e)
inhibiting growth in
a tumor cell. The tumor cell may be any type of tumor cell, such as a leukemia
cell. Other
types of cells include, for example, a bladder cancer cell, a breast cancer
cell, a lung cancer
cell, a colon cancer cell, a prostate cancer cell, a liver cancer cell, a
pancreatic cancer cell, a
stomach cancer cell, a testicular cancer cell, a brain cancer cell, an ovarian
cancer cell, a
lymphatic cancer cell, a skin cancer cell, a brain cancer cell, a bone cancer
cell, or a soft
tissue cancer cell.
Combination treatment therapy is also contemplated by the present disclosure.
For
example, regarding methods of treating cancer in a subject, comprising
administering to the
subject a pharmaceutically effective amount of a compound of the present
disclosure, the
method may further comprise a treatment selected from the group consisting of
administering
a pharmaceutically effective amount of a second drug, radiotherapy, gene
therapy, and
surgery. Such methods may further comprise (1) contacting a tumor cell with
the compound
prior to contacting the tumor cell with the second drug, (2) contacting a
tumor cell with the
second drug prior to contacting the tumor cell with the compound, or (3)
contacting a tumor
cell with the compound and the second drug at the same time. The second drug
may, in
certain embodiments, be an antibiotic, anti-inflammatory, anti-neoplastic,
anti-proliferative,
anti-viral, immunomodulatory, or immunosuppressive. The second drug may be an
alkylating agent, androgen receptor modulator, cytoskeletal disruptor,
estrogen receptor
modulator, histone-deacetylase inhibitor, HMG-CoA reductase inhibitor, prenyl-
protein
transferase inhibitor, retinoid receptor modulator, topoisomerase inhibitor,
or tyrosine kinase
inhibitor. In certain embodiments, the second drug is 5-azacitidine, 5-
fluorouracil, 9-cis-
retinoic acid, actinomycin D, alitretinoin, all-trans-retinoic acid,
annamycin, axitinib,
belinostat, bevacizumab, bexarotene, bosutinib, busulfan, capecitabine,
carboplatin,
carmustine, CD437, cediranib, cetuximab, chlorambucil, cisplatin,
cyclophosphamide,
cytarabine, dacarbazine, dasatinib, daunorubicin, decitabine, docetaxel,
dolastatin-10,
doxifluridine, doxorubicin, doxorubicin, epirubicin, erlotinib, etoposide,
etoposide, gefitinib,
gemcitabine, gemtuzumab ozogamicin, hexamethylmelamine, idarubicin,
ifosfamide,
imatinib, irinotecan, isotretinoin, ixabepilone, lapatinib, LBH589, lomustine,
mechlorethamine, melphalan, mercaptopurine, methotrexate, mitomycin,
mitoxantrone, MS-
275, neratinib, nilotinib, nitrosourea, oxaliplatin, paclitaxel, plicamycin,
procarbazine,
semaxanib, semustine, sodium butyrate, sodium phenylacetate, streptozotocin,
suberoylanilide hydroxamic acid, sunitinib, tamoxifen, teniposide, thiopeta,
tioguanine,
32
CA 02721837 2015-07-30
topotecan, TRAIL, trastuzumab, tretinoin, trichostatin A, valproic acid,
valrubicin,
vandetanib, vinblastine, vincristine, vindesine, or vinorelbine.
Methods of treating or preventing a disease with an inflammatory component in
a
subject, comprising administering to the subject a phaimaceutically effective
amount of a
compound of the present disclosure are also contemplated. The disease may be,
for example,
lupus or rheumatoid arthritis. The disease may be an inflammatory bowel
disease, such as
Crohn's disease or ulcerative colitis. The disease with an inflammatory
component may be a
cardiovascular disease. The disease with an inflammatory component may be
diabetes, such
as type 1 or type 2 diabetes. Compounds of the present disclosure may also be
used to treat
.1 0 complications associated with diabetes. Such complications are well-
known in the art and
include, for example, obesity, hypertension, atherosclerosis, coronary heart
disease, stroke,
peripheral vascular disease, hypertension, nephropathy, ncuropathy,
myonecrosis, retinopathy
and metabolic syndrome (syndrome X). The disease with an inflammatory
component may
be a skin disease, such as psoriasis, acne, or atopic dermatitis.
Administration of a compound
I 5 of the present disclosure in treatment methods of such skin diseases
may be, for example,
topical or oral.
The disease with an inflammatory component may be metabolic syndrome (syndrome
X). A patient having this syndrome is characterized as having three or more
symptoms
selected from the following group of five symptoms: (1) abdominal obesity; (2)
20 hypeftriglyceridemia; (3) low high-density lipoprotein cholesterol
(HDL); (4) high blood
pressure; and (5) elevated fasting glucose, which may be in the range
characteristic of Type 2
diabetes if the patient is also diabetic. Each of these symptoms is defined in
the Third Report
of the National Cholesterol Education Program Expert Panel on Detection,
Evaluation and
Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel 111, or
ATP III),
25 National Institutes of Health, 2001, NIH Publication No. 01-3670.
Patients with metabolic syndrome, whether or not they have or develop overt
diabetes mellitus, have an increased risk of developing the macrovascular and
microvascular
complications that are listed above that occur with type 2 diabetes, such as
atherosclerosis and
coronary heart disease.
30
Another general method of the present disclosure entails a method of treating
or
preventing a cardiovascular disease in a subject, comprising administering to
the subject a
pharmaceutically effective amount of a compound of the present disclosure.
The
cardiovascular disease may be, for example, atherosclerosis, cardiomyopathy,
congenital
33
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
heart disease, congestive heart failure, myocarditis, rheumatic heart disease,
valve disease,
coronary artery disease, endocarditis, or myocardial infarction. Combination
therapy is also
contemplated for such methods. For example, such methods may further comprise
administering a pharmaceutically effective amount of a second drug. The second
drug may
be, for example, a cholesterol lowering drug, an anti-hyperlipidemic, a
calcium channel
blocker, an anti-hypertensive, or an HMG-CoA reductase inhibitor. Non-limiting
examples
of second drugs include amlodipine, aspirin, ezetimibe, felodipine,
lacidipine, lercanidipine,
nicardipine, nifedipine, nimodipine, nisoldipine or nitrendipine. Other non-
limiting examples
of second drugs include atenolol, bucindolol, carvedilol, clonidine,
doxazosin, indoramin,
labetalol, methyldopa, metoprolol, nadolol, oxprenolol, phenoxybenzamine,
phentolamine,
pindolol, prazosin, propranolol, terazosin, timolol or tolazoline. The second
drug may be, for
example, a statin, such as atorvastatin, cerivastatin, fluvastatin,
lovastatin, mevastatin,
pitavastatin, pravastatin, rosuvastatin or simvastatin.
Methods of treating or preventing a neurodegenerative disease in a subject,
comprising administering to the subject a pharmaceutically effective amount of
a compound
of the present disclosure are also contemplated. The neurodegenerative disease
may, for
example, be selected from the group consisting of Parkinson's disease,
Alzheimer's disease,
multiple sclerosis (MS), Huntington's disease and amyotrophic lateral
sclerosis. In particular
embodiments, the neurodegenerative disease is Alzheimer's disease.
In particular
embodiments, the neurodegenerative disease is MS, such as primary progressive,
relapsing-
remitting secondary progressive or progressive relapsing MS. The subject may
be, for
example, a primate. The subject may be a human.
In particular embodiments of methods of treating or preventing a
neurodegenerative
disease in a subject, comprising administering to the subject a
pharmaceutically effective
amount of a compound of the present disclosure, the treatment suppresses the
demyelination
of neurons in the subject's brain or spinal cord. In certain embodiments, the
treatment
suppresses inflammatory demyelination. In certain embodiments, the treatment
suppresses
the transection of neuron axons in the subject's brain or spinal cord. In
certain embodiments,
the treatment suppresses the transection of neurites in the subject's brain or
spinal cord. In
certain embodiments, the treatment suppresses neuronal apoptosis in the
subject's brain or
spinal cord. In certain embodiments, the treatment stimulates the
remyelination of neuron
axons in the subject's brain or spinal cord. In certain embodiments, the
treatment restores lost
function after an MS attack. In certain embodiments, the treatment prevents a
new MS
34
CA 02721837 2015-07-30
attack. In certain embodiments, the treatment prevents a disability resulting
from an MS
attack.
One general aspect of the present disclosure contemplates a method of treating
or
preventing a disorder characterized by overexpression of iNOS genes in a
subject, comprising
administering to the subject a pharmaceutically effective amount of a compound
of the
present disclosure.
Another general aspect of the present disclosure contemplates a method of
inhibiting
1FN-7-induced nitric oxide production in cells of a subject, comprising
administering to said
subject a pharmaceutically effective amount of a compound of the present
disclosure.
Yet another general method of the present disclosure contemplates a method of
treating or preventing a disorder characterized by overexpression of COX-2
genes in a
subject, comprising administering to the subject a pharmaceutically effective
amount of
compound of the present disclosure.
Methods of treating renal/kidney disease (RKD) in a subject, comprising
administering to the subject a pharmaceutically effective amount of a compound
of the
present disclosure are also contemplated. See U.S. Patent Application
Publication
No. 2009-0326063.
The RKD may result from, for example, a
toxic insult. The toxic insult may result from, for example, an imaging agent
or a drug. The
drug may be a chemotherapeutic, for example.
The RKD may result from
ischemia/reperfusion injury, in certain embodiments. In certain embodiments,
the RKD
results from diabetes or hypertension. The RKD may result from an autoimmune
disease.
The RKD may be further defined as chronic RKD, or acute RKD.
In certain methods of treating renal/kidney disease (RKD) in a subject,
comprising
administering to the subject a pharmaceutically effective amount of a compound
of the
present disclosure, the subject has undergone or is undergoing dialysis. In
certain
embodiments, the subject has undergone or is a candidate to undergo kidney
transplant. The
subject may be a primate. The primate may be a human. The subject in this or
any other
method may be, for example, a cow, horse, dog, cat, pig, mouse, rat or guinea
pig.
Also contemplated by the present disclosure is a method for improving
glomerular
filtration rate or creatinine clearance in a subject, comprising administering
to the subject a
pharmaceutically effective amount of a compound of the present disclosure.
In some embodiments, the invention provides compounds useful for preventing
and/or
treating diseases or disorders whose pathology involves oxidative stress,
inflammation, and/or
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
dysregulation of inflammatory signaling pathways. In some variations, the
diseases or
disorders can be characterized by overexpression of inducible nitric oxide
synthase (iNOS)
and/or inducible cyclooxygenase (COX-2) in affected tissues. In some
variations, the
diseases or disorders can be characterized by overproduction of reactive
oxygen species
(ROS) or reactive nitrogen species (RNS) such as superoxide, hydrogen
peroxide, nitric oxide
or peroxynitrite in affected tissues. In some variations, the disease or
disorder is
characterized by excessive production of inflammatory cytokines or other
inflammation-
related proteins such as TNFa, IL-6, IL-1, IL-8, ICAM-1, VCAM-1, and VEGF.
Such
diseases or disorders may, in some embodiments, involve undesirable
proliferation of certain
cells, as in the case of cancer (e.g., solid tumors, leukemias, myelomas,
lymphomas, and other
cancers), fibrosis associated with organ failure, or excessive scarring. Non
limiting examples
of the disease or disorder include: lupus, rheumatoid arthritis, juvenile-
onset diabetes,
multiple sclerosis, psoriasis, and Crohn's disease. Further non-limiting
examples include
cardiovascular diseases, such as atherosclerosis, heart failure, myocardial
infarction, acute
coronary syndrome, restenosis following vascular surgery, hypertension, and
vasculitis;
neurodegenerative or neuromuscular diseases such as Alzheimer's disease,
Parkinson's
disease, Huntington's disease, ALS, and muscular dystrophy; neurological
disorders such as
epilepsy and dystonia; neuropsychiatric conditions such as major depression,
bipolar disorder,
post-traumatic stress disorder, schizophrenia, anorexia nervosa, ADHD, and
autism-spectrum
disorders; retinal diseases such as macular degeneration, diabetic
retinopathy, glaucoma, and
retinitis; chronic and acute pain syndromes, including inflammatory and
neuropathic pain;
hearing loss and tinnitus; diabetes and complications of diabetes, including
metabolic
syndrome, diabetic nephropathy, diabetic neuropathy and diabetic ulcers;
respiratory diseases
such as asthma, chronic obstructive pulmonary disease, acute respiratory
distress syndrome,
and cystic fibrosis; inflammatory bowel diseases; osteoporosis,
osteoarthritis, and other
degenerative conditions of bone and cartilage; acute or chronic organ failure,
including renal
failure, liver failure (including cirrhosis and hepatitis), and pancreatitis;
ischemia-reperfusion
injury associated with thrombotic or hemorrhagic stroke, subarachnoid
hemorrhage, cerebral
vasospasm, myocardial infarction, shock, or trauma; complications of organ or
tissue
transplantation including acute or chronic transplant failure or rejection and
graft-versus-host
disease; skin diseases including atopic dermatitis and acne; sepsis and septic
shock; excessive
inflammation associated with infection, including respiratory inflammation
associated with
36
CA 02721837 2015-07-30
influenza and upper respiratory infections; mucositis associated with cancer
therapy,
including radiation therapy or chemotherapy; and severe burns.
Methods of synthesizing compounds of the present disclosure are also
contemplated.
In particular embodiments, such methods can comprise a method of making a
target
compound defined of the formula:
0 ISO
Ra
NC
10,4poo
0
H
wherein Ra is alkoxy(c1.4), comprising reacting a compound of the formula:
0
Ra
NC Ispo
0
with an oxidizing agent under a set of conditions to form the target compound.
Kits are also contemplated by the present disclosure, such as a kit
comprising: a
compound of the present disclosure; and instructions which comprise one or
more forms of
information selected from the group consisting of indicating a disease state
for which the
compound is to be administered, storage information for the compound, dosing
information
and instructions regarding how to administer the compound. The kit may
comprise a
________________________________________ compound of the present disclosure in
a multiple dose fot tti.
Other objects, features and advantages of the present disclosure will become
apparent
from the following detailed description.
Note that simply because a particular compound is ascribed to
one particular generic formula doesn't mean that it cannot also belong to
another generic
formula.
37
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included
to
further demonstrate certain aspects of the present disclosure. The invention
may be better
understood by reference to one of these drawings in combination with the
detailed description
of specific embodiments presented herein.
FIGS. 1-8 and 32-34. Inhibition of NO Production. RAW264.7 macrophages were
pre-treated with DMSO or drugs at various concentrations (nM) for 2 hours,
then treated with
20 ng/ml IFNy for 24 hours. NO concentration in media was determined using a
Griess
reagent system; cell viability was determined using WST-1 reagent.
FIG. 9. Suppression of COX-2 Induction. RAW264.7 cells were pre-treated for 2
hours with indicated compounds and subsequently stimulated with 10 ng/ml IFNy
for an
additional 24 hours. COX-2 protein levels were assayed by immunoblotting.
Actin was used
as a loading control. RTA 402 and RTA 404 refer to comparison compounds 402
and 404
(see Example 1).
FIGS. 10-12. Inhibition of IL-6 Induced STAT3 Phosphorylation. HeLa cells
were treated with the indicated compounds and concentrations for 6 hours and
subsequently
stimulated with 20 ng/ml IL-6 for 15 minutes. Phosphorylated STAT3 and total
STAT3
levels were assayed by immunoblotting. Compounds 402-52 and 402-53 are
comparison
compounds (see Example 1).
FIG. 13. Suppression of IL-6 Induced STAT3 Phosphorylation. HeLa cells were
treated with DMSO or the indicated compounds at 2 ilM for 6 hours and
subsequently
stimulated with 20 ng/ml IL-6 for 15 minutes. Phosphorylated STAT3 and total
STAT3
levels were assayed by immunoblotting. Compounds 402-54, 402-55 and 402-56 are
comparison compounds (see Example 1).
FIG. 14. Inhibition of TNFa-induced bcBa degradation. HeLa cells were treated
with indicated compounds and concentrations for 6 hours and subsequently
stimulated with
20 ng/ml TNFa for 15 minutes. Lysates were analyzed with antibodies against
IKBa and
actin.
FIGS. 15 and 16. Inhibition of NFicB activation. HeLa cells were transfected
with
pNF-KB-Luc (inducible) and pRL-TK (constitutive) reporter plasmids. Twenty-
four hours
later cells were pre-treated with the indicated compounds for 2 hours. DMSO
served as a
38
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
vehicle control. Following pre-treatment, cells were stimulated with 20 ng/ml
TNFa for 3
hours. Reporter activity was measured using DualGlo luciferase reporter assay
and pNF-KB
luciferase activity was normalized against pRL-TK luciferase activity. Fold-
induction of
mean luciferase activity relative to unstimulated (-TNFa) samples is shown.
Error bars
represent the SD of the mean of 6 samples.
FIGS. 17- 20. Induction of HO-1. MDA-MB-435 human melanoma cells were
treated with vehicle (DMSO) or the indicated compounds and concentrations for
16 hours.
HO-1 mRNA levels were quantified using qPCR and were normalized relative to a
DMSO-
treated sample run in parallel. Values are averages of duplicate wells.
FIG. 21. Induction of HO-1, TrxR1 and y-GCS. MDA-MB-435 human melanoma
cells were treated with vehicle (DMSO) or the indicated compounds and
concentrations for
16 hours. HO-1, thioredoxin reductase-1 (TrxR1), and y-glutamylcysteine
synthetase (y-
GCS) mRNA levels were quantified using qPCR and were normalized relative to a
DMSO-
treated sample run in parallel. Values are averages of duplicate wells.
FIG. 22. Induction of TrxR1. MDA-MB-435 human melanoma cells were treated
with vehicle (DMSO) or the indicated compounds and concentrations for 16
hours.
Thioredoxin reductase-1 (TrxR1) mRNA levels were quantified using qPCR and
were
normalized relative to a DMSO-treated sample run in parallel. Values are
averages of
duplicate wells. Compounds 401, 402-19 and 402-53 are comparison compounds
(see
Example 1). Comparison with the results of FIG. 25 demonstrates that higher
concentrations
of 402-02 and 404-02 are required to approach effects seen with the
unsaturated counterpart
compounds 402 and 404.
FIG. 23. Induction of y-GCS. MDA-MB-435 human melanoma cells were treated
with vehicle (DMSO) or the indicated compounds and concentrations for 16
hours. y-
glutamylcysteine synthetase (y-GCS) mRNA levels were quantified using qPCR and
were
normalized relative to a DMSO-treated sample run in parallel. Values are
averages of
duplicate wells. Comparison with the results of FIG. 26 demonstrates that
higher
concentrations of 402-02 and 404-02 are required to approach effects seen with
the
unsaturated counterpart compounds 402 and 404.
FIG. 24. Induction of Ferritin Heavy Chain. MDA-MB-435 human melanoma
cells were treated with vehicle (DMSO) or the indicated compounds and
concentrations for
39
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
16 hours. Ferritin heavy chain mRNA levels were quantified using qPCR and were
normalized relative to a DMSO-treated sample run in parallel. Values are
averages of
duplicate wells. Comparison with the results of FIG. 27 demonstrates that
higher
concentrations of 402-02 and 404-02 are required to approach effects seen with
the
unsaturated counterpart compounds 402 and 404.
FIG. 25. Induction of TrxR1. MDA-MB-435 human melanoma cells were treated
with vehicle (DMSO) or the indicated compounds and concentrations for 16
hours.
Thioredoxin reductase-1 (TrxR1) mRNA levels were quantified using qPCR and
were
normalized relative to a DMSO-treated sample run in parallel. Values are
averages of
duplicate wells. Comparison with the results of FIG. 22 demonstrates that
higher
concentrations of 402-02 and 404-02 are required to approach effects seen with
the
unsaturated counterpart compounds 402 and 404.
FIG. 26. Induction of 'y-GCS. MDA-MB-435 human melanoma cells were treated
with vehicle (DMSO) or the indicated compounds and concentrations for 16
hours. y-
glutamylcysteine synthetase (y-GCS) mRNA levels were quantified using qPCR and
were
normalized relative to a DMSO-treated sample run in parallel. Values are
averages of
duplicate wells. Comparison with the results of FIG. 23 demonstrates that
higher
concentrations of 402-02 and 404-02 are required to approach effects seen with
the
unsaturated counterpart compounds 402 and 404.
FIG. 27. Induction of Ferritin Heavy Chain. MDA-MB-435 human melanoma
cells were treated with vehicle (DMSO) or the indicated compounds and
concentrations for
16 hours. Ferritin heavy chain mRNA levels were quantified using qPCR and were
normalized relative to a DMSO-treated sample run in parallel. Values are
averages of
duplicate wells. Comparison with the results of FIG. 24 demonstrates that
higher
concentrations of 402-02 and 404-02 are required to approach effects seen with
the
unsaturated counterpart compounds 402 and 404.
FIG. 28 ¨ CDDO-TFEA (TP-500) Is Detected at Higher Levels in Mouse Brain
than CDDO-EA (TP-319). CD-1 mice were fed either 200 or 400 mg/kg diet of
either
TP-319 or TP-500 for 3.5 days, and TP levels in the brains of the mice were
analyzed by
LC/MS. The structures of TP-319 and TP-500 are shown below.
CA 02721837 2010-10-15
WO 2009/129545 PCT/US2009/041170
FIGS. 29 A & B ¨ Weight Change Data from a Head to Head Tox Study of
Compounds 401 (FIG 29A) versus 401-02 (FIG. 29B). Compounds were assessed for
toxicity in mice in a 14-day study. Each compound was formulated in sesame oil
and
administered daily by oral gavage at doses of 10, 50, 100, or 250 mg/kg (n = 4
per group).
FIGS. 30 A & B ¨ Weight Change Data from a Head to Head Tox Study of
Compounds 402 (FIG 30A) versus 402-02 (FIG. 30B). Compounds were assessed for
toxicity in mice in a 14-day study. Each compound was formulated in sesame oil
and
administered daily by oral gavage at doses of 10, 50, 100, or 250 mg/kg (n = 4
per group).
FIGS. 31 A & B ¨ Weight Change Data from a Head to Head Tox Study of
Compounds 404 (FIG 31A) versus 404-02 (FIG. 31B). Compounds were assessed for
toxicity in mice in a 14-day study. Each compound was formulated in sesame oil
and
administered daily by oral gavage at doses of 10, 50, 100, or 250 mg/kg (n = 4
per group).
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
Disclosed herein are, for example, new compounds with antioxidant and anti-
inflammatory properties, methods for their manufacture, and methods for their
use, including
for the treatment and/or prevention of disease.
I. Definitions
As used herein, "hydrogen" means ¨H; "hydroxy" means ¨OH; "oxo" means =0;
"halo" means independently ¨F, ¨C1, ¨Br or ¨I; "amino" means ¨NH2 (see below
for
definitions of groups containing the term amino, e.g., alkylamino);
"hydroxyamino" means
¨NHOH; "nitro" means ¨NO2; imino means =NH (see below for definitions of
groups
containing the term imino, e.g., alkylamino); "cyano" means ¨CN; "azido" means
¨N3;
"mercapto" means ¨SH; "thio" means =S; "sulfonamido" means ¨NHS(0)2¨ (see
below for
definitions of groups containing the term sulfonamido, e.g.,
alkylsulfonamido); "sulfonyl"
means ¨S(0)2¨ (see below for definitions of groups containing the term
sulfonyl, e.g.,
alkylsulfonyl); and "sily1" means ¨SiH3 (see below for definitions of group(s)
containing the
term silyl, e.g., alkylsilyl).
For the groups below, the following parenthetical subscripts further define
the groups
as follows: "(Cn)" defines the exact number (n) of carbon atoms in the group.
"(Cn)" defines
the maximum number (n) of carbon atoms that can be in the group, with the
minimum
number of carbon atoms in such at least one, but otherwise as small as
possible for the group
41
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
in question. E.g., it is understood that the minimum number of carbon atoms in
the group
"alkenyl(c<8)" is 2. For example, "alkoxy(c<10)" designates those alkoxy
groups having from 1
to 10 carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any range
derivable therein (e.g., 3-
carbon atoms)). (Cn-n') defines both the minimum (n) and maximum number (n')
of
5
carbon atoms in the group. Similarly, "alkyl(c2_10)" designates those alkyl
groups having from
2 to 10 carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any range
derivable therein (e.g., 3-
10 carbon atoms)).
The term "alkyl" when used without the "substituted" modifier refers to a non-
aromatic monovalent group with a saturated carbon atom as the point of
attachment, a linear
10 or
branched, cyclo, cyclic or acyclic structure, no carbon-carbon double or
triple bonds, and
no atoms other than carbon and hydrogen. The groups, -CH3 (Me), -CH2CH3 (Et),
-CH2CH2CH3 (n-Pr), -CH(CH3)2 (iso-Pr), -CH(CH2)2 (cyclopropyl), -CH2CH2CH2CH3
(n-
Bu), -CH(CH3)CH2CH3 (sec-butyl), -CH2CH(CH3)2 (iso-butyl), -C(CH3)3 (tert-
butyl),
-CH2C(CH3)3 (neo-pentyl), cyclobutyl, cyclopentyl, cyclohexyl, and
cyclohexylmethyl are
non-limiting examples of alkyl groups. The term "substituted alkyl" refers to
a non-aromatic
monovalent group with a saturated carbon atom as the point of attachment, a
linear or
branched, cyclo, cyclic or acyclic structure, no carbon-carbon double or
triple bonds, and at
least one atom independently selected from the group consisting of N, 0, F,
Cl, Br, I, Si, P,
and S. The following groups are non-limiting examples of substituted alkyl
groups:
-CH2OH, -CH2C1, -CH2Br, -CH2SH, -CF3, -CH2CN, -CH2C(0)H, -CH2C(0)0H,
-CH2C(0)0CH3, -CH2C(0)NH2, -CH2C(0)NHCH3, -CH2C(0)CH3, -CH2OCH3,
-CH2OCH2CF3, -CH20C(0)CH3, -CH2NH2, -CH2NHCH3, -CH2N(CH3)2, -CH2CH2C1,
-CH2CH2OH, -CH2CF3, -CH2CH20C(0)CH3, -CH2CH2NHCO2C(CH3)3, and
-CH2Si(CH3)3.
The term "alkanediyl" when used without the "substituted" modifier refers to a
non-
aromatic divalent group, wherein the alkanediyl group is attached with two a-
bonds, with one
or two saturated carbon atom(s) as the point(s) of attachment, a linear or
branched, cyclo,
cyclic or acyclic structure, no carbon-carbon double or triple bonds, and no
atoms other than
carbon and hydrogen. The groups, -CH2- (methylene), -CH2CH2-, -CH2C(CH3)2CH2-,
-,55.1-
-CH2CH2CH2-, and - , are non-limiting examples of alkanediyl groups. The
term "substituted alkanediyl" refers to a non-aromatic monovalent group,
wherein the
alkynediyl group is attached with two a-bonds, with one or two saturated
carbon atom(s) as
42
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
the point(s) of attachment, a linear or branched, cyclo, cyclic or acyclic
structure, no carbon-
carbon double or triple bonds, and at least one atom independently selected
from the group
consisting of N, 0, F, Cl, Br, I, Si, P, and S. The following groups are non-
limiting examples
of substituted alkanediyl groups: ¨CH(F)¨, ¨CF2¨, ¨CH(C1)¨, ¨CH(OH)¨,
¨CH(OCH3)¨,
and ¨CH2CH(C1)¨.
The term "alkenyl" when used without the "substituted" modifier refers to a
monovalent group with a nonaromatic carbon atom as the point of attachment, a
linear or
branched, cyclo, cyclic or acyclic structure, at least one nonaromatic carbon-
carbon double
bond, no carbon-carbon triple bonds, and no atoms other than carbon and
hydrogen. Non-
limiting examples of alkenyl groups include: ¨CH=CH2 (vinyl), ¨CH=CHCH35
¨CH=CHCH2CH3, ¨CH2CH=CH2 (allyl), ¨CH2CH=CHCH3, and ¨CH=CH¨C6H5. The term
"substituted alkenyl" refers to a monovalent group with a nonaromatic carbon
atom as the
point of attachment, at least one nonaromatic carbon-carbon double bond, no
carbon-carbon
triple bonds, a linear or branched, cyclo, cyclic or acyclic structure, and at
least one atom
independently selected from the group consisting of N, 0, F, Cl, Br, I, Si, P,
and S. The
groups, ¨CH=CHF, ¨CH=CHC1 and ¨CH=CHBr, are non-limiting examples of
substituted
alkenyl groups.
The term "alkenediyl" when used without the "substituted" modifier refers to a
non-
aromatic divalent group, wherein the alkenediyl group is attached with two a-
bonds, with two
carbon atoms as points of attachment, a linear or branched, cyclo, cyclic or
acyclic structure,
at least one nonaromatic carbon-carbon double bond, no carbon-carbon triple
bonds, and no
atoms other than carbon and hydrogen. The groups, ¨CH=CH¨, ¨CH=C(CH3)CH2-5
.ss
¨CH=CHCH2¨, and '.
, are non-limiting examples of alkenediyl groups. The
term "substituted alkenediyl" refers to a non-aromatic divalent group, wherein
the alkenediyl
group is attached with two a-bonds, with two carbon atoms as points of
attachment, a linear
or branched, cyclo, cyclic or acyclic structure, at least one nonaromatic
carbon-carbon double
bond, no carbon-carbon triple bonds, and at least one atom independently
selected from the
group consisting of N, 0, F, Cl, Br, I, Si, P, and S. The following groups are
non-limiting
examples of substituted alkenediyl groups: ¨CF=CH¨, ¨C(OH)=CH¨, and
¨CH2CH=C(C1)¨.
The term "alkynyl" when used without the "substituted" modifier refers to a
monovalent group with a nonaromatic carbon atom as the point of attachment, a
linear or
branched, cyclo, cyclic or acyclic structure, at least one carbon-carbon
triple bond, and no
43
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
atoms other than carbon and hydrogen. The groups, ¨CCH, ¨CCCH3, ¨CCC6H5 and
¨CH2CCCH3, are non-limiting examples of alkynyl groups. The term "substituted
alkynyl"
refers to a monovalent group with a nonaromatic carbon atom as the point of
attachment and
at least one carbon-carbon triple bond, a linear or branched, cyclo, cyclic or
acyclic structure,
and at least one atom independently selected from the group consisting of N,
0, F, Cl, Br, I,
Si, P, and S. The group, ¨CCSi(CH3)3, is a non-limiting example of a
substituted alkynyl
group.
The term "alkynediyl" when used without the "substituted" modifier refers to a
non-
aromatic divalent group, wherein the alkynediyl group is attached with two a-
bonds, with two
carbon atoms as points of attachment, a linear or branched, cyclo, cyclic or
acyclic structure,
at least one carbon-carbon triple bond, and no atoms other than carbon and
hydrogen. The
groups, ¨CC¨, ¨CCCH2¨, and ¨CCCH(CH3)¨ are non-limiting examples of alkynediyl
groups. The term "substituted alkynediyl" refers to a non-aromatic divalent
group, wherein
the alkynediyl group is attached with two a-bonds, with two carbon atoms as
points of
attachment, a linear or branched, cyclo, cyclic or acyclic structure, at least
one carbon-carbon
triple bond, and at least one atom independently selected from the group
consisting of N, 0,
F, Cl, Br, I, Si, P, and S. The groups ¨CCCFH¨ and ¨CCHCH(C1)¨ are non-
limiting
examples of substituted alkynediyl groups.
The term "aryl" when used without the "substituted" modifier refers to a
monovalent
group with an aromatic carbon atom as the point of attachment, said carbon
atom forming
part of a six-membered aromatic ring structure wherein the ring atoms are all
carbon, and
wherein the monovalent group consists of no atoms other than carbon and
hydrogen. Non-
limiting examples of aryl groups include phenyl (Ph), methylphenyl,
(dimethyl)phenyl,
¨C6H4CH2CH3 (ethylphenyl),
C6H4CH2CH2CH3 (propylphenyl) 5 C 6H4CH(CH3 )2 5
¨C6H4CH(CH2)2, ¨C6H3(CH3)CH2CH3 (methylethylphenyl), ¨C6H4CH=CH2
(vinylphenyl),
¨C6H4CH=CHCH3, ¨C6H4CCH, ¨C6H4CCCH3, naphthyl, and the monovalent group
derived from biphenyl. The term "substituted aryl" refers to a monovalent
group with an
aromatic carbon atom as the point of attachment, said carbon atom forming part
of a six-
membered aromatic ring structure wherein the ring atoms are all carbon, and
wherein the
monovalent group further has at least one atom independently selected from the
group
consisting of N, 0, F, Cl, Br, I, Si, P, and S. Non-limiting examples of
substituted aryl
groups include the groups: ¨C6H4F, ¨C6H4C1, C6H4Br, ¨C6H41, C6H4OH, C6H4OCH3,
¨C6H4OCH2CH3, C6H40C(0)CH3, C6H4NH2, C6H4NHCH3, ¨C6H4N(CH3)25
44
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
C6H4CH2OH, C6H4CH20C(0)CH3, C6H4CH2NH2, C6H4CF3, C6H4CN, C6H4CHO,
C6H4CHO, C6H4C(0)CH3, C6H4C(0)C6H5, C6H4CO2H, C6H4CO2CH3, C6H4CONH2,
-C6H4CONHCH3, and ¨C6H4CON(CH3)2.
The term "arenediyl" when used without the "substituted" modifier refers to a
divalent
group, wherein the arenediyl group is attached with two a-bonds, with two
aromatic carbon
atoms as points of attachment, said carbon atoms forming part of one or more
six-membered
aromatic ring structure(s) wherein the ring atoms are all carbon, and wherein
the monovalent
group consists of no atoms other than carbon and hydrogen. Non-limiting
examples of
arenediyl groups include:
..f.j., H3C
¨/
. 1¨ 5 ¨/ = ¨1 . SO ' and -1 41 /¨
5 5 .
The term "substituted arenediyl" refers to a divalent group, wherein the
arenediyl group is
attached with two a-bonds, with two aromatic carbon atoms as points of
attachment, said
carbon atoms forming part of one or more six-membered aromatic rings
structure(s), wherein
the ring atoms are all carbon, and wherein the divalent group further has at
least one atom
independently selected from the group consisting of N, 0, F, Cl, Br, I, Si, P,
and S.
The term "aralkyl" when used without the "substituted" modifier refers to the
monovalent group ¨alkanediyl¨aryl, in which the terms alkanediyl and aryl are
each used in a
manner consistent with the definitions provided above. Non-limiting examples
of aralkyls
are: phenylmethyl (benzyl, Bn), 1-phenyl-ethyl, 2-phenyl-ethyl, indenyl and
2,3-dihydro-
indenyl, provided that indenyl and 2,3-dihydro-indenyl are only examples of
aralkyl in so far
as the point of attachment in each case is one of the saturated carbon atoms.
When the term
"aralkyl" is used with the "substituted" modifier, either one or both the
alkanediyl and the
aryl is substituted. Non-limiting examples of substituted aralkyls are: (3-
chloropheny1)-
methyl, 2-oxo-2-phenyl-ethyl (phenylcarbonylmethyl), 2-chloro-2-phenyl-ethyl,
chromanyl
where the point of attachment is one of the saturated carbon atoms, and
tetrahydroquinolinyl
where the point of attachment is one of the saturated atoms.
The term "heteroaryl" when used without the "substituted" modifier refers to a
monovalent group with an aromatic carbon atom or nitrogen atom as the point of
attachment,
said carbon atom or nitrogen atom forming part of an aromatic ring structure
wherein at least
one of the ring atoms is nitrogen, oxygen or sulfur, and wherein the
monovalent group
consists of no atoms other than carbon, hydrogen, aromatic nitrogen, aromatic
oxygen and
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
aromatic sulfur. Non-limiting examples of aryl groups include acridinyl,
furanyl,
imidazoimidazolyl, imidazopyrazolyl, imidazopyridinyl, imidazopyrimidinyl,
indolyl,
indazolinyl, methylpyridyl, oxazolyl, phenylimidazolyl, pyridyl, pyrrolyl,
pyrimidyl,
pyrazinyl, quinolyl, quinazolyl, quinoxalinyl, tetrahydroquinolinyl, thienyl,
triazinyl,
pyrrolopyridinyl, pyrrolopyrimidinyl, pyrrolopyrazinyl, pyrrolotriazinyl,
pyrroloimidazolyl,
chromenyl (where the point of attachment is one of the aromatic atoms), and
chromanyl
(where the point of attachment is one of the aromatic atoms). The term
"substituted
heteroaryl" refers to a monovalent group with an aromatic carbon atom or
nitrogen atom as
the point of attachment, said carbon atom or nitrogen atom forming part of an
aromatic ring
structure wherein at least one of the ring atoms is nitrogen, oxygen or
sulfur, and wherein the
monovalent group further has at least one atom independently selected from the
group
consisting of non-aromatic nitrogen, non-aromatic oxygen, non aromatic sulfur
F, Cl, Br, I,
Si, and P.
The term "heteroarenediyl" when used without the "substituted" modifier refers
to a
divalent group, wherein the heteroarenediyl group is attached with two a-
bonds, with an
aromatic carbon atom or nitrogen atom as the point of attachment, said carbon
atom or
nitrogen atom two aromatic atoms as points of attachment, said carbon atoms
forming part of
one or more six-membered aromatic ring structure(s) wherein the ring atoms are
all carbon,
and wherein the monovalent group consists of no atoms other than carbon and
hydrogen.
Non-limiting examples of heteroarenediyl groups include:
-1 /
N--\-
__I
2-1-
H and --i- N
, .
The term "substituted heteroarenediyl" refers to a divalent group, wherein the
heteroarenediyl
group is attached with two a-bonds, with two aromatic carbon atoms as points
of attachment,
said carbon atoms forming part of one or more six-membered aromatic rings
structure(s),
wherein the ring atoms are all carbon, and wherein the divalent group further
has at least one
atom independently selected from the group consisting of N, 0, F, Cl, Br, I,
Si, P, and S.
The term "heteroaralkyl" when used without the "substituted" modifier refers
to the
monovalent group ¨alkanediyl¨heteroaryl, in which the terms alkanediyl and
heteroaryl are
each used in a manner consistent with the definitions provided above. Non-
limiting examples
of aralkyls are: pyridylmethyl, and thienylmethyl. When the term
"heteroaralkyl" is used
46
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
with the "substituted" modifier, either one or both the alkanediyl and the
heteroaryl is
substituted.
The term "acyl" when used without the "substituted" modifier refers to a
monovalent
group with a carbon atom of a carbonyl group as the point of attachment,
further having a
linear or branched, cyclo, cyclic or acyclic structure, further having no
additional atoms that
are not carbon or hydrogen, beyond the oxygen atom of the carbonyl group. The
groups,
-CHO, -C(0)CH3 (acetyl, Ac), -C(0)CH2CH3, -C(0)CH2CH2CH3, -C(0)CH(CH3)2,
-C(0)CH(CH2)2, -C(0)C6H5, -C(0)C6H4CH3, -C(0)C6H4CH2CH3, -00C6H3(CH3)2, and
-C(0)CH2C6H5, are non-limiting examples of acyl groups. The term "acyl"
therefore
encompasses, but is not limited to groups sometimes referred to as "alkyl
carbonyl" and "aryl
carbonyl" groups. The term "substituted acyl" refers to a monovalent group
with a carbon
atom of a carbonyl group as the point of attachment, further having a linear
or branched,
cyclo, cyclic or acyclic structure, further having at least one atom, in
addition to the oxygen
of the carbonyl group, independently selected from the group consisting of N,
0, F, Cl, Br, I,
Si, P, and S. The groups, -C(0)CH2CF3, -CO2H (carboxyl), -CO2CH3
(methylcarboxyl),
-CO2CH2CH3, -CO2CH2CH2CH3, CO2C6H5, -CO2CH(CH3)2, -CO2CH(CH2)2, -C(0)NH2
(carbamoyl), -C(0)NHCH3, -C(0)NHCH2CH3, -CONHCH(CH3)2, -CONHCH(CH2)2,
-CON(CH3)2, -CONHCH2CF3, -CO-pyridyl, -CO-imidazoyl, and -C(0)N3, are non-
limiting examples of substituted acyl groups. The term "substituted acyl"
encompasses, but is
not limited to, "heteroaryl carbonyl" groups.
The term "alkylidene" when used without the "substituted" modifier refers to
the
divalent group =CRR', wherein the alkylidene group is attached with one a-bond
and one 7C-
bond, in which R and R' are independently hydrogen, alkyl, or R and R' are
taken together to
represent alkanediyl.
Non-limiting examples of alkylidene groups include: =CH2,
=CH(CH2CH3), and =C(CH3)2. The term "substituted alkylidene" refers to the
group =CRR',
wherein the alkylidene group is attached with one a-bond and one 7c-bond, in
which R and R'
are independently hydrogen, alkyl, substituted alkyl, or R and R' are taken
together to
represent a substituted alkanediyl, provided that either one of R and R' is a
substituted alkyl
or R and R' are taken together to represent a substituted alkanediyl.
The term "alkoxy" when used without the "substituted" modifier refers to the
group
-OR, in which R is an alkyl, as that term is defined above. Non-limiting
examples of alkoxy
groups include: -OCH3, -OCH2CH3, -OCH2CH2CH3, -OCH(CH3)2, -OCH(CH2)2,
-0-cyclopentyl, and -0-cyclohexyl. The term "substituted alkoxy" refers to the
group
47
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
¨OR, in which R is a substituted alkyl, as that term is defined above. For
example,
¨OCH2CF3 is a substituted alkoxy group.
Similarly, the terms "alkenyloxy", "alkynyloxy", "aryloxy", "aralkoxy",
"heteroaryloxy", "heteroaralkoxy" and "acyloxy", when used without the
"substituted"
modifier, refers to groups, defined as ¨OR, in which R is alkenyl, alkynyl,
aryl, aralkyl,
heteroaryl, heteroaralkyl and acyl, respectively, as those terms are defined
above. When any
of the terms alkenyloxy, alkynyloxy, aryloxy, aralkyloxy and acyloxy is
modified by
"substituted," it refers to the group ¨OR, in which R is substituted alkenyl,
alkynyl, aryl,
aralkyl, heteroaryl, heteroaralkyl and acyl, respectively.
The term "alkylamino" when used without the "substituted" modifier refers to
the
group ¨NHR, in which R is an alkyl, as that term is defined above. Non-
limiting examples of
alkylamino groups include: ¨NHCH3, ¨NHCH2CH3, ¨NHCH2CH2CH3, ¨NHCH(CH3)2,
¨NHCH(CH2)2, ¨NHCH2CH2CH2CH3, ¨NHCH(CH3)CH2CH3, ¨NHCH2CH(CH3)2,
¨NHC(CH3)3, ¨NH¨cyclopentyl, and ¨NH¨cyclohexyl. The term "substituted
alkylamino"
refers to the group ¨NHR, in which R is a substituted alkyl, as that term is
defined above.
For example, ¨NHCH2CF3 is a substituted alkylamino group.
The term "dialkylamino" when used without the "substituted" modifier refers to
the
group ¨NRR', in which R and R' can be the same or different alkyl groups, or R
and R' can be
taken together to represent an alkanediyl having two or more saturated carbon
atoms, at least
two of which are attached to the nitrogen atom. Non-limiting examples of
dialkylamino
groups include: ¨NHC(CH3)3, ¨N(CH3)CH2CH3, ¨N(CH2CH3)2, N-pyrrolidinyl, and N-
piperidinyl. The term "substituted dialkylamino" refers to the group ¨NRR', in
which R and
R' can be the same or different substituted alkyl groups, one of R or R' is an
alkyl and the
other is a substituted alkyl, or R and R' can be taken together to represent a
substituted
alkanediyl with two or more saturated carbon atoms, at least two of which are
attached to the
nitrogen atom.
The terms "alkoxyamino", "alkenylamino", "alkynylamino", "arylamino",
"aralkylamino", "heteroarylamino", "heteroaralkylamino", and
"alkylsulfonylamino" when
used without the "substituted" modifier, refers to groups, defined as ¨NHR, in
which R is
alkoxy, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl and
alkylsulfonyl,
respectively, as those terms are defined above. A non-limiting example of an
arylamino
group is ¨NHC6H5. When any of the terms alkoxyamino, alkenylamino,
alkynylamino,
arylamino, aralkylamino, heteroarylamino, heteroaralkylamino and
alkylsulfonylamino is
48
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
modified by "substituted," it refers to the group ¨NHR, in which R is
substituted alkoxy,
alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl and alkylsulfonyl,
respectively.
The term "amido" (acylamino), when used without the "substituted" modifier,
refers
to the group ¨NHR, in which R is acyl, as that term is defined above. A non-
limiting
example of an acylamino group is ¨NHC(0)CH3. When the term amido is used with
the
"substituted" modifier, it refers to groups, defined as ¨NHR, in which R is
substituted acyl, as
that term is defined above. The groups ¨NHC(0)0CH3 and ¨NHC(0)NHCH3 are non-
limiting examples of substituted amido groups.
The term "alkylimino" when used without the "substituted" modifier refers to
the
group =NR, wherein the alkylimino group is attached with one a-bond and one 7c-
bond, in
which R is an alkyl, as that term is defined above. Non-limiting examples of
alkylimino
groups include: =NCH3, =NCH2CH3 and =N¨cyclohexyl. The term "substituted
alkylimino"
refers to the group =NR, wherein the alkylimino group is attached with one a-
bond and one
7c-bond, in which R is a substituted alkyl, as that term is defined above. For
example,
=NCH2CF3 is a substituted alkylimino group.
Similarly, the terms "alkenylimino", "alkynylimino", "arylimino",
"aralkylimino",
"heteroarylimino", "heteroaralkylimino" and "acylimino", when used without the
"substituted" modifier, refers to groups, defined as =NR, wherein the
alkylimino group is
attached with one a-bond and one 7c-bond, in which R is alkenyl, alkynyl,
aryl, aralkyl,
heteroaryl, heteroaralkyl and acyl, respectively, as those terms are defined
above. When any
of the terms alkenylimino, alkynylimino, arylimino, aralkylimino and acylimino
is modified
by "substituted," it refers to the group =NR, wherein the alkylimino group is
attached with
one a-bond and one 7c-bond, in which R is substituted alkenyl, alkynyl, aryl,
aralkyl,
heteroaryl, heteroaralkyl and acyl, respectively.
The term "fluoroalkyl" when used without the "substituted" modifier refers to
an
alkyl, as that term is defined above, in which one or more fluorines have been
substituted for
hydrogens. The groups, ¨CH2F, ¨CF3, and ¨CH2CF3 are non-limiting examples of
fluoroalkyl groups. The term "substituted fluoroalkyl" refers to a non-
aromatic monovalent
group with a saturated carbon atom as the point of attachment, a linear or
branched, cyclo,
cyclic or acyclic structure, at least one fluorine atom, no carbon-carbon
double or triple
bonds, and at least one atom independently selected from the group consisting
of N, 0, Cl,
Br, I, Si, P, and S. The following group is a non-limiting example of a
substituted
fluoroalkyl: ¨CFHOH.
49
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
The term "alkylthio" when used without the "substituted" modifier refers to
the group
¨SR, in which R is an alkyl, as that term is defined above. Non-limiting
examples of
alkylthio groups include: ¨SCH3, ¨SCH2CH3, ¨SCH2CH2CH3, ¨SCH(CH3)2,
¨SCH(CH2)25
¨S¨cyclopentyl, and ¨S¨cyclohexyl. The term "substituted alkylthio" refers to
the group
¨SR, in which R is a substituted alkyl, as that term is defined above. For
example,
¨SCH2CF3 is a substituted alkylthio group.
Similarly, the terms "alkenylthio", "alkynylthio", "arylthio", "aralkylthio",
"heteroarylthio", "heteroaralkylthio", and "acylthio", when used without the
"substituted"
modifier, refers to groups, defined as ¨SR, in which R is alkenyl, alkynyl,
aryl, aralkyl,
heteroaryl, heteroaralkyl and acyl, respectively, as those terms are defined
above. When any
of the terms alkenylthio, alkynylthio, arylthio, aralkylthio, heteroarylthio,
heteroaralkylthio,
and acylthio is modified by "substituted," it refers to the group ¨SR, in
which R is substituted
alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl and acyl,
respectively.
The term "thioacyl" when used without the "substituted" modifier refers to a
monovalent group with a carbon atom of a thiocarbonyl group as the point of
attachment,
further having a linear or branched, cyclo, cyclic or acyclic structure,
further having no
additional atoms that are not carbon or hydrogen, beyond the sulfur atom of
the carbonyl
group. The groups, ¨CHS, ¨C(S)CH3, ¨C(S)CH2CH3, ¨C(S)CH2CH2CH3, ¨C(S)CH(CH3)25
¨C(S)CH(CH2)2, ¨C(S)C6H5, ¨C(S)C6H4CH3, ¨C(S)C6H4CH2CH3, ¨C(S)C6H3(CH3)2, and
¨C(S)CH2C6H5, are non-limiting examples of thioacyl groups. The term
"thioacyl" therefore
encompasses, but is not limited to, groups sometimes referred to as "alkyl
thiocarbonyl" and
"aryl thiocarbonyl" groups. The term "substituted thioacyl" refers to a
radical with a carbon
atom as the point of attachment, the carbon atom being part of a thiocarbonyl
group, further
having a linear or branched, cyclo, cyclic or acyclic structure, further
having at least one
atom, in addition to the sulfur atom of the carbonyl group, independently
selected from the
group consisting of N, 0, F, Cl, Br, I, Si, P, and S. The groups, ¨C(S)CH2CF3,
¨C(S)02H,
¨C(S)OCH3, ¨C(S)OCH2CH3, ¨C(S)OCH2CH2CH3, ¨C(S)0C6H5, ¨C(S)OCH(CH3)25
¨C(S)OCH(CH2)2, ¨C(S)NH2, and ¨C(S)NHCH3, are non-limiting examples of
substituted
thioacyl groups. The term "substituted thioacyl" encompasses, but is not
limited to,
"heteroaryl thiocarbonyl" groups.
The term "alkylsulfonyl" when used without the "substituted" modifier refers
to the
group ¨S(0)2R, in which R is an alkyl, as that term is defined above. Non-
limiting examples
of alkylsulfonyl groups include: S(0)2CH3, S(0)2CH2CH3, S(0)2CH2CH2CH3,
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
-S(0)2CH(CH3)25 -S(0)2CH(CH2)25 ¨S(0)2¨cyclopentyl, and ¨S(0)2¨cyclohexyl. The
term
"substituted alkylsulfonyl" refers to the group ¨S(0)2R, in which R is a
substituted alkyl, as
that term is defined above. For example, ¨S(0)2CH2CF3 is a substituted
alkylsulfonyl group.
Similarly, the terms "alkenylsulfonyl", "alkynylsulfonyl", "arylsulfonyl",
"aralkylsulfonyl", "heteroarylsulfonyl", and "heteroaralkylsulfonyl" when used
without the
"substituted" modifier, refers to groups, defined as ¨S(0)2R, in which R is
alkenyl, alkynyl,
aryl, aralkyl, heteroaryl, and heteroaralkyl, respectively, as those terms are
defined above.
When any of the terms alkenylsulfonyl, alkynylsulfonyl, arylsulfonyl,
aralkylsulfonyl,
heteroarylsulfonyl, and heteroaralkylsulfonyl is modified by "substituted," it
refers to the
group ¨S(0)2R, in which R is substituted alkenyl, alkynyl, aryl, aralkyl,
heteroaryl and
heteroaralkyl, respectively.
The term "alkylammonium" when used without the "substituted" modifier refers
to a
group, defined as ¨NH2R', ¨NHRR'+, or ¨NRR'R"+, in which R, R' and R" are the
same or
different alkyl groups, or any combination of two of R, R' and R" can be taken
together to
represent an alkanediyl. Non-limiting examples of alkylammonium cation groups
include:
¨NH2(CH3)', ¨NH2(CH2CH3)+, ¨NH2(CH2CH2CH3)+, ¨NH(CH3)2', ¨NH(CH2CH3)2'5
¨NH(CH2CH2CH3)2 -N(C143)3 -N(CH3)(CH2CH3)2
-N(CH3)2(CH2CH3)
-NH2C(CH3)3 ¨NH(cyc1openty1)2', and ¨NH2(cyc1ohexy1)'.
The term "substituted
alkylammonium" refers ¨NH2R', ¨NHRR'+, or ¨NRR'R"+, in which at least one of
R, R and
R" is a substituted alkyl or two of R, R' and R" can be taken together to
represent a
substituted alkanediyl. When more than one of R, R' and R" is a substituted
alkyl, they can
be the same of different. Any of R, R' and R" that are not either substituted
alkyl or
substituted alkanediyl, can be either alkyl, either the same or different, or
can be taken
together to represent a alkanediyl with two or more carbon atoms, at least two
of which are
attached to the nitrogen atom shown in the formula.
The term "alkylsulfonium" when used without the "substituted" modifier refers
to the
group ¨SRR'', in which R and R' can be the same or different alkyl groups, or
R and R' can
be taken together to represent an alkanediyl. Non-limiting examples of
alkylsulfonium
groups include: ¨SH(CH3)', ¨SH(CH2CH3)', ¨SH(CH2CH2CH3)', ¨S(CH3)2
¨S(CH2CH3)2'5 ¨S(CH2CH2CH3)2'5 ¨SH(cyclopentyl)', and ¨SH(cyclohexyl)'. The
term
"substituted alkylsulfonium" refers to the group ¨SRR'', in which R and R' can
be the same
or different substituted alkyl groups, one of R or R' is an alkyl and the
other is a substituted
51
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
alkyl, or R and R' can be taken together to represent a substituted
alkanediyl. For example,
-SH(CH2CF3) is a substituted alkylsulfonium group.
The term "alkylsily1" when used without the "substituted" modifier refers to a
monovalent group, defined as -SiH2R, -SiHRR', or -SiRR'R", in which R, R' and
R" can be
the same or different alkyl groups, or any combination of two of R, R' and R"
can be taken
together to represent an alkanediyl. The groups, -SiH2CH3, -SiH(CH3)2, -
Si(CH3)3 and
-Si(CH3)2C(CH3)3, are non-limiting examples of unsubstituted alkylsilyl
groups. The term
"substituted alkylsily1" refers -SiH2R, -SiHRR', or -SiRR'R", in which at
least one of R, R'
and R" is a substituted alkyl or two of R, R' and R" can be taken together to
represent a
substituted alkanediyl. When more than one of R, R' and R" is a substituted
alkyl, they can
be the same of different. Any of R, R' and R" that are not either substituted
alkyl or
substituted alkanediyl, can be either alkyl, either the same or different, or
can be taken
together to represent a alkanediyl with two or more saturated carbon atoms, at
least two of
which are attached to the silicon atom.
In addition, atoms making up the compounds of the present disclosure are
intended to
include all isotopic forms of such atoms. Isotopes, as used herein, include
those atoms having
the same atomic number but different mass numbers. By way of general example
and
without limitation, isotopes of hydrogen include tritium and deuterium, and
isotopes of
carbon include 13C and 14C. Similarly, it is contemplated that one or more
carbon atom(s) of
a compound of the present disclosure may be replaced by a silicon atom(s).
Furthermore, it is
contemplated that one or more oxygen atom(s) of a compound of the present
disclosure may
be replaced by a sulfur or selenium atom(s).
A compound having a formula that is represented with a dashed bond is intended
to
include the formulae optionally having zero, one or more double bonds. Thus,
for example,
---.
r
I., ,..1
the structure - includes the structures 0, O, 1, ISI and 10.
As will be understood by a person of skill in the art, no one such ring atom
forms part of more
than one double bond.
Any undefined valency on an atom of a structure shown in this application
implicitly
represents a hydrogen atom bonded to the atom.
A ring structure shown with an unconnected "R" group, indicates that any
implicit
hydrogen atom on that ring can be replaced with that R group. In the case of a
divalent R
52
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
group (e.g., oxo, imino, thio, alkylidene, etc.), any pair of implicit
hydrogen atoms attached to
one atom of that ring can be replaced by that R group. This concept is as
exemplified below:
0 -..../
...........R
represents
0
<01.õ...51 ca R M
,
,
or
As used herein, a "chiral auxiliary" refers to a removable chiral group that
is capable
of influencing the stereoselectivity of a reaction. Persons of skill in the
art are familiar with
such compounds, and many are commercially available.
The use of the word "a" or "an," when used in conjunction with the term
"comprising" in the claims and/or the specification may mean "one," but it is
also consistent
with the meaning of "one or more," "at least one," and "one or more than one."
Throughout this application, the term "about" is used to indicate that a value
includes
the inherent variation of error for the device, the method being employed to
determine the
value, or the variation that exists among the study subjects.
The terms "comprise," "have" and "include" are open-ended linking verbs. Any
forms or tenses of one or more of these verbs, such as "comprises,"
"comprising," "has,"
"having," "includes" and "including," are also open-ended. For example, any
method that
"comprises," "has" or "includes" one or more steps is not limited to
possessing only those
one or more steps and also covers other unlisted steps.
The term "effective," as that term is used in the specification and/or claims,
means
adequate to accomplish a desired, expected, or intended result.
The term "hydrate" when used as a modifier to a compound means that the
compound
has less than one (e.g., hemihydrate), one (e.g., monohydrate), or more than
one (e.g.,
dihydrate) water molecules associated with each compound molecule, such as in
solid forms
of the compound.
As used herein, the term "IC50" refers to an inhibitory dose which is 50% of
the
maximum response obtained.
53
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
An "isomer" of a first compound is a separate compound in which each molecule
contains the same constituent atoms as the first compound, but where the
configuration of
those atoms in three dimensions differs.
As used herein, the term "patient" or "subject" refers to a living mammalian
organism,
such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig,
or transgenic
species thereof. In certain embodiments, the patient or subject is a primate.
Non-limiting
examples of human subjects are adults, juveniles, infants and fetuses.
"Pharmaceutically acceptable" means that which is useful in preparing a
pharmaceutical composition that is generally safe, non-toxic and neither
biologically nor
otherwise undesirable and includes that which is acceptable for veterinary use
as well as
human pharmaceutical use.
"Pharmaceutically acceptable salts" means salts of compounds of the present
disclosure which are pharmaceutically acceptable, as defined above, and which
possess the
desired pharmacological activity. Such salts include acid addition salts
formed with inorganic
acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,
phosphoric acid,
and the like; or with organic acids such as 1,2-ethanedisulfonic acid, 2-
hydroxyethanesulfonic
acid, 2-naphthalenesulfonic acid, 3 -phenylpropionic
acid,
4,4'-methylenebis (3 -hydroxy-2 -ene-1 -carboxylic acid), 4-methylbicyclo [2
.2 .2] o ct-2-ene-
1-carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids,
aliphatic sulfuric acids,
aromatic sulfuric acids, benzenesulfonic acid, benzoic acid, camphorsulfonic
acid, carbonic
acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic
acid, fumaric
acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid,
heptanoic acid, hexanoic
acid, hydroxynaphthoic acid, lactic acid, laurylsulfuric acid, maleic acid,
malic acid, malonic
acid, mandelic acid, methanesulfonic acid, muconic acid, o-(4-
hydroxybenzoyl)benzoic acid,
oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substituted alkanoic acids,
propionic acid,
p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic
acid, tartaric acid,
tertiarybutylacetic acid, trimethylacetic acid, and the like. Pharmaceutically
acceptable salts
also include base addition salts which may be formed when acidic protons
present are capable
of reacting with inorganic or organic bases. Acceptable inorganic bases
include sodium
hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and
calcium
hydroxide. Acceptable organic bases include ethanolamine, diethanolamine,
triethanolamine,
tromethamine, N-methylglucamine and the like. It should be recognized that the
particular
anion or cation forming a part of any salt of this invention is not critical,
so long as the salt, as
54
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
a whole, is pharmacologically acceptable. Additional examples of
pharmaceutically
acceptable salts and their methods of preparation and use are presented in
Handbook of
Pharmaceutical Salts: Properties, and Use (2002).
As used herein, "predominantly one enantiomer" means that a compound contains
at
least about 85% of one enantiomer, or more preferably at least about 90% of
one enantiomer,
or even more preferably at least about 95% of one enantiomer, or most
preferably at least
about 99% of one enantiomer. Similarly, the phrase "substantially free from
other optical
isomers" means that the composition contains at most about 15% of another
enantiomer or
diastereomer, more preferably at most about 10% of another enantiomer or
diastereomer,
even more preferably at most about 5% of another enantiomer or diastereomer,
and most
preferably at most about 1% of another enantiomer or diastereomer.
"Prevention" or "preventing" includes: (1) inhibiting the onset of a disease
in a subject
or patient which may be at risk and/or predisposed to the disease but does not
yet experience
or display any or all of the pathology or symptomatology of the disease,
and/or (2) slowing
the onset of the pathology or symptomatology of a disease in a subject or
patient which may
be at risk and/or predisposed to the disease but does not yet experience or
display any or all of
the pathology or symptomatology of the disease.
"Prodrug" means a compound that is convertible in vivo metabolically into an
inhibitor according to the present disclosure. The prodrug itself may or may
not also have
activity with respect to a given target protein. For example, a compound
comprising a
hydroxy group may be administered as an ester that is converted by hydrolysis
in vivo to the
hydroxy compound. Suitable esters that may be converted in vivo into hydroxy
compounds
include acetates, citrates, lactates, phosphates, tartrates, malonates,
oxalates, salicylates,
propionates, succinates, fumarates, maleates, methy1ene-bis-I3-
hydroxynaphthoate, gentisates,
isethionates, di-p-toluoyltartrates, methanesulfonates, ethanesulfonates,
benzenesulfonates,
p-toluenesulfonates, cyclohexylsulfamates, quinates, esters of amino acids,
and the like.
Similarly, a compound comprising an amine group may be administered as an
amide that is
converted by hydrolysis in vivo to the amine compound.
The term "saturated" when referring to an atom means that the atom is
connected to
other atoms only by means of single bonds.
A "stereoisomer" or "optical isomer" is an isomer of a given compound in which
the
same atoms are bonded to the same other atoms, but where the configuration of
those atoms
in three dimensions differs. "Enantiomers" are stereoisomers of a given
compound that are
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
mirror images of each other, like left and right hands. "Diastereomers" are
stereoisomers of a
given compound that are not enantiomers.
The invention contemplates that for any stereocenter or axis of chirality for
which
stereochemistry has not been defined, that stereocenter or axis of chirality
can be present in
its R form, S form, or as a mixture of the R and S forms, including racemic
and non-racemic
mixtures.
"Substituent convertible to hydrogen in vivo" means any group that is
convertible to a
hydrogen atom by enzymological or chemical means including, but not limited
to, hydrolysis
and hydrogenolysis. Examples include acyl groups, groups having an oxycarbonyl
group,
amino acid residues, peptide residues, o-nitrophenylsulfenyl, trimethylsilyl,
tetrahydro-
pyranyl, diphenylphosphinyl, hydroxy or alkoxy substituents on imino groups,
and the like.
Examples of acyl groups include formyl, acetyl, trifluoroacetyl, and the like.
Examples of
groups having an oxycarbonyl group include ethoxycarbonyl, tert-butoxycarbonyl
(¨C(0)0C(CH3)3), benzyloxycarbonyl, p-methoxybenzyloxycarbonyl,
vinyloxycarbonyl, 13-
(p-toluenesulfonyl)ethoxycarbonyl, and the like. Suitable amino acid residues
include, but
are not limited to, residues of Gly (glycine), Ala (alanine), Arg (arginine),
Asn (asparagine),
Asp (aspartic acid), Cys (cysteine), Glu (glutamic acid), His (histidine), Ile
(isoleucine), Leu
(leucine), Lys (lysine), Met (methionine), Phe (phenylalanine), Pro (proline),
Ser (serine), Thr
(threonine), Trp (tryptophan), Tyr (tyrosine), Val (valine), Nva (norvaline),
Hse
(homoserine), 4-Hyp (4-hydroxyproline), 5-Hyl (5-hydroxylysine), Om
(ornithine) and 13-Ala.
Examples of suitable amino acid residues also include amino acid residues that
are protected
with a protecting group. Examples of suitable protecting groups include those
typically
employed in peptide synthesis, including acyl groups (such as formyl and
acetyl),
arylmethyloxycarbonyl groups (such as benzyloxycarbonyl and p-
nitrobenzyloxycarbonyl),
tert-butoxycarbonyl groups (¨C(0)0C(CH3)3), and the like. Suitable peptide
residues
include peptide residues comprising two to five, and optionally amino acid
residues. The
residues of these amino acids or peptides can be present in stereochemical
configurations of
the D-form, the L-form or mixtures thereof In addition, the amino acid or
peptide residue
may have an asymmetric carbon atom. Examples of suitable amino acid residues
having an
asymmetric carbon atom include residues of Ala, Leu, Phe, Trp, Nva, Val, Met,
Ser, Lys, Thr
and Tyr. Peptide residues having an asymmetric carbon atom include peptide
residues having
one or more constituent amino acid residues having an asymmetric carbon atom.
Examples
of suitable amino acid protecting groups include those typically employed in
peptide
56
CA 02721837 2015-07-30
synthesis, including acyl groups (such as formyl and acetyl),
arylmethyloxycarbonyl groups
(such as benzyloxycarbonyl and p-nitrobenzyloxycarbonyl), tert-butoxycarbonyl
groups
(¨C(0)0C(CH3)3), and the like. Other examples of substituents "convertible to
hydrogen in
vivo" include reductively eliminable hydrogenolyzable groups.
Examples of suitable
reductively eliminable hydrogenolyzable groups include, but are not limited
to, arylsulfonyl
groups (such as o-toluenesulfonyl); methyl groups substituted with phenyl or
benzyloxy (such
as benzyl, trityl and benzyloxymethyl); arylmethoxycarbonyl groups (such as
benzyloxycarbonyl and o-methoxy-benzyloxycarbonyl); and haloethoxycarbonyl
groups
(such as P,3,[3-trichloroethoxycarbonyl and P-iodoethoxycarbony1).
"Therapeutically effective amount" or "pharmaceutically effective amount"
means
that amount which, when administered to a subject or patient for treating a
disease, is
sufficient to effect such treatment for the disease.
"Treatment" or "treating" includes (1) inhibiting a disease in a subject or
patient
experiencing or displaying the pathology or symptomatology of the disease
(e.g., arresting
further development of the pathology and/or symptomatology), (2) ameliorating
a disease in a
subject or patient that is experiencing or displaying the pathology or
symptomatology of the
disease (e.g., reversing the pathology and/or symptomatology), and/or (3)
effecting any
measurable decrease in a disease in a subject or patient that is experiencing
or displaying the
pathology or symptomatology of the disease.
As used herein, the term "water soluble" means that the compound dissolves in
water
at least to the extent of 0.010 mole/liter or is classified as soluble
according to literature
precedence.
Other abbreviations used herein are as follows: DMSO, dimethyl sulfoxide; NO,
nitric
oxide; iNOS, inducible nitric oxide synthase; COX-2, cyclooxygenase-2; NGF,
nerve growth
factor; IBMX, isobutylmethylxanthine; FBS, fetal bovine serum; GPDH, glycerol
3-
phosphate dehydrogenase; RXR, retinoid X receptor; TGF-43, transforming growth
factor-{3;
IFNy or IFN-y, interferon-y; LPS, bacterial endotoxic lipopolysaccharide; TNFa
or TNF-a,
tumor necrosis factor-a; IL-113, interleukin-113; GAPDH, glyceraldehyde-3-
phosphate
dehydrogenase; MTT, 344,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide; TCA,
trichloroacetic acid; HO-1, inducible heme oxygenase.
The above definitions supersede any conflicting definition.
The fact that certain terms are defined, however, should
not be considered as indicative that any term that is undefined is indefinite.
Rather, all terms
57
CA 02721837 2015-07-30
used are believed to describe the invention in terms such that one of ordinary
skill can
appreciate the scope and practice the present disclosure.
11. Synthetic Methods
Compounds of the present disclosure may be made using the methods outlined in
the
Examples section (Example 2 and 3). These methods can be further modified and
optimized
using the principles and techniques of organic chemistry as applied by a
person skilled in the
art. Such principles and techniques are taught, for example, in March's
Advanced Organic
Chemistry: Reactions, Mechanisms, and Structure (2007).
III. Biological Activity of Oleanolic Acid Derivatives
Biological activity results, both in vivo and in vitro are provided throughout
the
present disclosure. These include: inhibition of NO Production, suppression of
COX-2
induction, inhibition of 1L-6 induced STAT3 phosphorylation, suppression of 1L-
6 induced
STAT3 phosphorylation, inhibition of TNFa-induced 1kBa degradation, inhibition
of NFKB
activation, induction of HO-1, Nrf2induction of HO-1, TrxR1 and y-GCS,
induction of
TrxR1, induction of 'y-GCS, induction of ferritin heavy chain, induction of
TrxR1, induction
of -y-GCS, induction of ferritin heavy chain, and various in vivo toxicity
studies. See figures
and figure descriptions. Suppression of NO production and induction of Nrf2
induction
results can be respectively summarized as shown Tables la and lb, below.
Further results,
including toxicity studies, are provided in the Examples section.
Table la. Suppression of IFNy-Induced NO Production.
RAW264.7 (20 ng/ml IFN7)
Compound 1D(s) MW iNOS suppr.
NO 1050 WST-1 1050
WB
63101 / 402-02 / dh402 507.70 ¨12 nM 200 nM >90%
63102 / 404-02 / dh404 574.72 ¨45 nM > 200 nM
63250 / 402-46 509.70 > 200 nM > 200 nM
63197 / 402-48 512.72 > 200 nM > 200 nM
63195 / 402-49 509.72 > 200 nM > 200 nM
63196 / 402-51 / dh401 493.68 ¨75 nM > 200 nM
58
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
RAW264.7 (20 ng/m1IFNy)
Compound ID(s) MW iNOS
suppr.
NO ICso WST-1 IC50
WB
63252 / 402-57 474.68 -5 nM > 200 nM
63205 / 402-59 492.69 -25 nM > 200 nM
63206 / 402-64 477.68 -10 nM > 200 nM
63207 / 402-66 509.72 -50 nM > 200 nM
63219 / 402-78 509.72 -150 nM > 200 nM
63229 517.71 > 200nM > 200nM
63230 531.74 -50 nM > 200 nM
63227 576.73 > 200nM > 200nM
63219 509.72 -150 nM > 200 nM
63223 576.73 > 200nM > 200nM
63237 572.70 -80 nM > 200 nM
63268 576.75 > 200 nM > 200 nM
63274 547.81 - 70 nM > 200 nM
63289 525.73 > 200 nM > 200 nM
63295 522.73 - 20 nM > 200 nM
63296 522.73 - 200 nM > 200 nM
63308 491.70 - 25 nM > 200 nM
63323 583.80 - 40 nM See FIG. 33
63325 520.75 - 50 nM See FIG. 32
63326 552.79 - 50 nM See FIG. 34
59
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
Table lb. Induction of HO-1, TrxR1 and 7-GCS in Human Melanoma Cells.
Nrf2 target gene induction in MDA-MB-435 cells
Compound
400 nM* 250 nM**
Code
HO-1 TrxR1 y-GC S HO-1 NQ01 y-GCS
63101
4 47 53 3 2 5
(dh402)
63102
3 2
4.5
(dh404)
63196
1 48 26
(dh401)
63252 5 3
63205 1.7 1.7
3.5
63206 2.5 1.8
4.5
63207 1.2 1.6
3.1
63237 3 2.3
6.4
Blank entry: Not determined.
* Data expressed as a percent of induction observed for 402 (see below for
structure).
** Data expressed as fold induction above DMSO control.
In certain embodiments, the compounds of the present disclosure are capable of
crossing the blood brain barrier and achieving therapeutically effective
concentrations in the
brain. They may therefore be used to treat neurodegenerative diseases, brain
cancer and other
inflammatory conditions affecting the central nervous system. For example, 404-
02 has been
shown to cross the blood-brain barrier and achieve high concentrations in the
central nervous
system tissue following oral dosing. Like the other compounds of the present
disclosure, it
promotes the resolution of innate and adaptive immune-mediated inflammation by
restoring
redox homeostasis in inflamed tissues. It is a potent inducer of the
antioxidant transcription
factor Nrf2 and inhibitor of the pro-oxidant/pro-inflammatory transcription
factors NF-KB
and the STATs. These biological pathways are implicated in a wide variety of
diseases,
including autoimmune conditions and several neurodegenerative diseases.
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
IV. Improved Rodent Toxicology
In certain embodiments, the invention provides compounds possessing low
toxicity in
rodents. In some cases, toxicity in rodents has been observed in preclinical
studies with some
analogues containing a carbon-carbon double bond in the C-ring, including 402
and 401.
Compounds having a saturated C-ring, in contrast, have consistently shown low
toxicity in
rodents. Predictably low rodent toxicity provides an advantage since high
rodent toxicity can
be a significant complication in conducting preclinical studies required for
development and
registration of therapeutic compounds for use in humans or non-human animals.
Illustrations
of this effect are provided below.
0 0
C-ring O C-ring O
C H3 CH3
NC es NC 0*
0 0
dh402 402
For example, an initial study (Example 6) was performed in Sprague Dawley rats
using both 402 and 402-02 and showed that 402-02 was less toxic. In a further
study
(Example 7), six compounds (401, 402, 404, 401-2, 402-2, and 404-2) were
assessed for
toxicity in mice in a 14-day study. At higher doses (above 10 mg/kg/day) both
401 and 402
caused at least 50% mortality, while 404 was non-toxic. In contrast, no
mortality was
observed in the 402-2 and 404-2 groups and only the highest dose of 401-02
caused any
lethality (Table 5). Body weight measurements (FIGS. 29-31) were consistent
with the
mortality observations. Notably, the two highest doses of 401 and 402 were
lethal within 4
days, in contrast to the effects of 401-2 and 402-2.
V. Diseases Associated with Inflammation and/or Oxidative Stress
Inflammation is a biological process that provides resistance to infectious or
parasitic
organisms and the repair of damaged tissue. Inflammation is commonly
characterized by
localized vasodilation, redness, swelling, and pain, the recruitment of
leukocytes to the site of
infection or injury, production of inflammatory cytokines such as TNF-a and IL-
1, and
production of reactive oxygen or nitrogen species such as hydrogen peroxide,
superoxide and
peroxynitrite. In later stages of inflammation, tissue remodeling,
angiogenesis, and scar
61
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
formation (fibrosis) may occur as part of the wound healing process. Under
normal
circumstances, the inflammatory response is regulated and temporary and is
resolved in an
orchestrated fashion once the infection or injury has been dealt with
adequately. However,
acute inflammation can become excessive and life-threatening if regulatory
mechanisms fail.
Alternatively, inflammation can become chronic and cause cumulative tissue
damage or
systemic complications.
Many serious and intractable human diseases involve dysregulation of
inflammatory
processes, including diseases such as cancer, atherosclerosis, and diabetes,
which were not
traditionally viewed as inflammatory conditions. In the case of cancer, the
inflammatory
processes are associated with tumor formation, progression, metastasis, and
resistance to
therapy. Atherosclerosis, long viewed as a disorder of lipid metabolism, is
now understood to
be primarily an inflammatory condition, with activated macrophages playing an
important
role in the formation and eventual rupture of atherosclerotic plaques.
Activation of
inflammatory signaling pathways has also been shown to play a role in the
development of
insulin resistance, as well as in the peripheral tissue damage associated with
diabetic
hyperglycemia. Excessive production of reactive oxygen species and reactive
nitrogen
species such as superoxide, hydrogen peroxide, nitric oxide, and peroxynitrite
is a hallmark of
inflammatory conditions. Evidence of dysregulated peroxynitrite production has
been
reported in a wide variety of diseases (Szabo et al., 2007; Schulz et al.,
2008; Forstermann,
2006; Pall, 2007).
Autoimmune diseases such as rheumatoid arthritis, lupus, psoriasis, and
multiple
sclerosis involve inappropriate and chronic activation of inflammatory
processes in affected
tissues, arising from dysfunction of self vs. non-self recognition and
response mechanisms in
the immune system. In neurodegenerative diseases such as Alzheimer's and
Parkinson's
diseases, neural damage is correlated with activation of microglia and
elevated levels of pro-
inflammatory proteins such as inducible nitric oxide synthase (iNOS). Chronic
organ failure
such as renal failure, heart failure, and chronic obstructive pulmonary
disease is closely
associated with the presence of chronic oxidative stress and inflammation,
leading to the
development of fibrosis and eventual loss of organ function.
Many other disorders involve oxidative stress and inflammation in affected
tissues,
including inflammatory bowel disease; inflammatory skin diseases; mucositis
related to
radiation therapy and chemotherapy; eye diseases such as uveitis, glaucoma,
macular
degeneration, and various forms of retinopathy; transplant failure and
rejection; ischemia-
62
CA 02721837 2015-07-30
reperfusion injury; chronic pain; degenerative conditions of the bones and
joints including
osteoarthritis and osteoporosis; asthma and cystic fibrosis; seizure
disorders; and
neuropsychiatric conditions including schizophrenia, depression, bipolar
disorder, post-
traumatic stress disorder, attention deficit disorders, autism-spectrum
disorders, and eating
disorders such as anorexia nervosa. Dysregulation of inflammatory signaling
pathways is
believed to be a major factor in the pathology of muscle wasting diseases
including muscular
dystrophy and various forms of cachexia.
A variety of life-threatening acute disorders also involve dysregulated
inflammatory
signaling, including acute organ failure involving the pancreas, kidneys,
liver, or lungs,
myocardial infarction or acute coronary syndrome, stroke, septic shock,
trauma, severe burns,
and anaphylaxis.
Many complications of infectious diseases also involve dysregulation of
inflammatory
responses. Although an inflammatory response can kill invading pathogens, an
excessive
inflammatory response can also be quite destructive and in some cases can be a
primary
source of damage in infected tissues. Furthermore, an excessive inflammatory
response can
also lead to systemic complications due to overproduction of inflammatory
cytokines such as
TNF-a and IL-1. This is believed to be a factor in mortality arising from
severe influenza,
severe acute respiratory syndrome, and sepsis.
The aberrant or excessive expression of either iNOS or cyclooxygenase-2 (COX-
2)
has been implicated in the pathogenesis of many disease processes. For
example, it is clear
that NO is a potent mutagen (Tamir and Tannebaum, 1996), and that nitric oxide
can also
activate COX-2 (Salvemini et al., 1994). Furthermore, there is a marked
increase in iNOS in
rat colon tumors induced by the carcinogen, azoxymethane (Takahashi et al.,
1997). A series
of synthetic triterpenoid analogs of oleanolic acid have been shown to be
powerful inhibitors
of cellular inflammatory processes, such as the induction by IFN-y of
inducible nitric oxide
synthase (iNOS) and of COX-2 in mouse macrophages. See Honda et al. (2000a);
Honda et
al. (2000b), and Honda et al. (2002).
In one aspect, compounds of the invention are characterized by their ability
to inhibit
the production of nitric oxide in macrophage-derived RAW 264.7 cells induced
by exposure
to 7-interferon. They are further characterized by their ability to induce the
expression of
antioxidant proteins such as NQ01 and reduce the expression of pro-
inflammatory proteins
such as COX-2 and inducible nitric oxide synthase (iNOS). These properties are
relevant to
the treatment of a wide array of diseases involving oxidative stress and
dysregulation of
63
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
inflammatory processes including cancer, mucositis resulting from radiation
therapy or
chemotherapy, autoimmune diseases, cardiovascular diseases including
atherosclerosis,
ischemia-reperfusion injury, acute and chronic organ failure including renal
failure and heart
failure, respiratory diseases, diabetes and complications of diabetes, severe
allergies,
transplant rejection, graft-versus-host disease, neurodegenerative diseases,
diseases of the eye
and retina, acute and chronic pain, degenerative bone diseases including
osteoarthritis and
osteoporosis, inflammatory bowel diseases, dermatitis and other skin diseases,
sepsis, burns,
seizure disorders, and neuropsychiatric disorders.
Without being bound by theory, the activation of the antioxidant/anti-
inflammatory
Keapl/Nrf2/ARE pathway is believed to be implicated in both the anti-
inflammatory and
anti-carcinogenic properties of the present oleanolic acid derivatives.
In another aspect, compounds of the invention may be used for treating a
subject
having a condition caused by elevated levels of oxidative stress in one or
more tissues.
Oxidative stress results from abnormally high or prolonged levels of reactive
oxygen species
such as superoxide, hydrogen peroxide, nitric oxide, and peroxynitrite (formed
by the reaction
of nitric oxide and superoxide). The oxidative stress may be accompanied by
either acute or
chronic inflammation. The oxidative stress may be caused by mitochondrial
dysfunction, by
activation of immune cells such as macrophages and neutrophils, by acute
exposure to an
external agent such as ionizing radiation or a cytotoxic chemotherapy agent
(e.g.,
doxorubicin), by trauma or other acute tissue injury, by ischemia/reperfusion,
by poor
circulation or anemia, by localized or systemic hypoxia or hyperoxia, by
elevated levels of
inflammatory cytokines and other inflammation-related proteins, and/or by
other abnormal
physiological states such as hyperglycemia or hypoglycemia.
In animal models of many such conditions, stimulating expression of inducible
heme
oxygenase (H0-1), a target gene of the Nrf2 pathway, has been shown to have a
significant
therapeutic effect including models of myocardial infarction, renal failure,
transplant failure
and rejection, stroke, cardiovascular disease, and autoimmune disease (e.g.,
Sacerdoti et al.,
2005; Abraham & Kappas, 2005; Bach, 2006; Araujo et al., 2003; Liu et al.,
2006; Ishikawa
et al., 2001; Kruger et al., 2006; Satoh et al., 2006; Zhou et al., 2005;
Morse and Choi, 2005;
Morse and Choi, 2002). This enzyme breaks free heme down into iron, carbon
monoxide
(CO), and biliverdin (which is subsequently converted to the potent
antioxidant molecule,
bilirubin).
64
CA 02721837 2015-07-30
In another aspect, compounds of this invention may be used in preventing or
treating
tissue damage or organ failure, acute and chronic, resulting from oxidative
stress exacerbated
by inflammation. Examples of diseases that fall in this category include:
heart failure, liver
failure, transplant failure and rejection, renal failure, pancreatitis,
fibrotic lung diseases
(cystic fibrosis and COPD, among others), diabetes (including complications),
atherosclerosis, ischemia-reperfusion injury, glaucoma, stroke, autoimmune
disease, autism,
macular degeneration, and muscular dystrophy. For example, in the case of
autism, studies
suggest that increased oxidative stress in the central nervous system may
contribute to the
development of the disease (Chauhan and Chauhan, 2006).
Evidence also links oxidative stress and inflammation to the development and
pathology of many other disorders of the central nervous system, including
psychiatric
disorders such as psychosis, major depression, and bipolar disorder; seizure
disorders such as
epilepsy; pain and sensory syndromes such as migraine, neuropathic pain or
tinnitus; and
behavioral syndromes such as the attention deficit disorders. See, e.g.,
Dickerson et al., 2007;
Hanson et al., 2005; Kendall-Tackett, 2007; Lencz et al., 2007; Dudhgaonkar et
al., 2006;
Lee et al., 2007; Morris et al., 2002; Ruster et al., 2005; McIver et al.,
2005; Sarchielli et al.,
2006; Kawakami et al., 2006; Ross et al., 2003.
For example, elevated levels of inflammatory cytokines, including TNF,
interferon-7,
and IL-6, are associated with major mental illness (Dickerson et al., 2007).
Microglial
activation has also been linked to major mental illness.
Therefore, downregulating
inflammatory cytokines and inhibiting excessive activation of microglia could
be beneficial in
patients with schizophrenia, major depression, bipolar disorder, autism-
spectrum disorders,
and other neuropsychiatric disorders.
Accordingly, in pathologies involving oxidative stress alone or oxidative
stress
exacerbated by inflammation, treatment may comprise administcring to a subject
a
therapeutically effective amount of a compound of this invention, such as
those described
above or throughout this specification. Treatment may be administered
preventively, in
advance of a predictable state of oxidative stress (e.g., organ
transplantation or the
administration of radiation therapy to a cancer patient), or it may be
administered
therapeutically in settings involving established oxidative stress and
inflammation.
The compounds of the invention may be generally applied to the treatment of
inflammatory conditions, such as sepsis, dermatitis, autoimmune disease and
osteoarthritis.
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
In one aspect, the compounds of this invention may be used to treat
inflammatory pain and/or
neuropathic pain, for example, by inducing Nrf2 and/or inhibiting NF-KB.
In one aspect, the compounds of the invention may be used to function as
antioxidant
inflammation modulators (AIMs) having potent anti-inflammatory properties that
mimic the
biological activity of cyclopentenone prostaglandins (cyPGs). In one
embodiment, the
compounds of the invention may be used to control the production of pro-
inflammatory
cytokines by selectively targeting regulatory cysteine residues (RCRs) on
proteins that
regulate the transcriptional activity of redox-sensitive transcription
factors. Activation of
RCRs by cyPGs or AIMs has been shown to initiate a pro-resolution program in
which the
activity of the antioxidant and cytoprotective transcription factor Nrf2 is
potently induced,
and the activities of the pro-oxidant and pro-inflammatory transcription
factors NF-KB and
the STATs are suppressed. This increases the production of antioxidant and
reductive
molecules (e.g., NQ01, HO-1, SOD1, and/or y-GCS) and/or decreases oxidative
stress and
the production of pro-oxidant and pro-inflammatory molecules (e.g., iNOS, COX-
2, and/or
TNF-a).
In some embodiments, the compounds of the invention may be used in the
treatment
and prevention of diseases such as cancer, inflammation, Alzheimer's disease,
Parkinson's
disease, multiple sclerosis, autism, amyotrophic lateral sclerosis, autoimmune
diseases such
as rheumatoid arthritis, lupus, and MS, inflammatory bowel disease, all other
diseases whose
pathogenesis is believed to involve excessive production of either nitric
oxide or
prostaglandins, and pathologies involving oxidative stress alone or oxidative
stress
exacerbated by inflammation.
Another aspect of inflammation is the production of inflammatory
prostaglandins such
as prostaglandin E. These molecules promote vasodilation, plasma
extravasation, localized
pain, elevated temperature, and other symptoms of inflammation. The inducible
form of the
enzyme COX-2 is associated with their production, and high levels of COX-2 are
found in
inflamed tissues. Consequently, inhibition of COX-2 may relieve many symptoms
of
inflammation and a number of important anti-inflammatory drugs (e.g.,
ibuprofen and
celecoxib) act by inhibiting COX-2 activity. Recent research, however, has
demonstrated that
a class of cyclopentenone prostaglandins (cyPGs) (e.g., 15-deoxy prostaglandin
J2, a.k.a.
PGJ2) plays a role in stimulating the orchestrated resolution of inflammation
(e.g., Rajakariar
et al., 2007). COX-2 is also associated with the production of cyclopentenone
prostaglandins.
Consequently, inhibition of COX-2 may interfere with the full resolution of
inflammation,
66
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
potentially promoting the persistence of activated immune cells in tissues and
leading to
chronic, "smoldering" inflammation. This effect may be responsible for the
increased
incidence of cardiovascular disease in patients using selective COX-2
inhibitors for long
periods of time.
In one aspect, the compounds of the invention may be used to control the
production
of pro-inflammatory cytokines within the cell by selectively activating
regulatory cysteine
residues (RCRs) on proteins that regulate the activity of redox-sensitive
transcription factors.
Activation of RCRs by cyPGs has been shown to initiate a pro-resolution
program in which the
activity of the antioxidant and cytoprotective transcription factor Nrf2 is
potently induced and
the activities of the pro-oxidant and pro-inflammatory transcription factors
NF-KB and the
STATs are suppressed. In some embodiments, this increases the production of
antioxidant and
reductive molecules (NQ01, HO-1, SOD1, y-GCS) and decreases oxidative stress
and the
production of pro-oxidant and pro-inflammatory molecules (iNOS, COX-2, TNF-a).
In some
embodiments, the compounds of this invention may cause the cells that host the
inflammatory
event to revert to a non-inflammatory state by promoting the resolution of
inflammation and
limiting excessive tissue damage to the host.
A. Cancer
Further, the compounds of the present disclosure may be used to induce
apoptosis in
tumor cells, to induce cell differentiation, to inhibit cancer cell
proliferation, to inhibit an
inflammatory response, and/or to function in a chemopreventative capacity. For
example, the
invention provides new compounds that have one or more of the following
properties: (1) an
ability to induce apoptosis and differentiate both malignant and non-malignant
cells, (2) an
activity at sub-micromolar or nanomolar levels as an inhibitor of
proliferation of many
malignant or premalignant cells, (3) an ability to suppress the de novo
synthesis of the
inflammatory enzyme inducible nitric oxide synthase (iNOS), (4) an ability to
inhibit NF-KB
activation, and (5) an ability to induce the expression of heme oxygenase-1
(H0-1).
The levels of iNOS and COX-2 are elevated in certain cancers and have been
implicated in carcinogenesis and COX-2 inhibitors have been shown to reduce
the incidence
of primary colonic adenomas in humans (Rostom et al., 2007; Brown and DuBois,
2005;
Crowel et al., 2003). iNOS is expressed in myeloid-derived suppressor cells
(MDSCs)
(Angulo et al., 2000) and COX-2 activity in cancer cells has been shown to
result in the
production of prostaglandin E2 (PGE2), which has been shown to induce the
expression of
67
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
arginase in MDSCs (Sinha et al., 2007). Arginase and iNOS are enzymes that
utilize L-
arginine as a substrate and produce L-ornithine and urea, and L-citrulline and
NO,
respectively. The depletion of arginine from the tumor microenvironment by
MDSCs,
combined with the production of NO and peroxynitrite has been shown to inhibit
proliferation
and induce apoptosis of T cells (Bronte et al., 2003). Inhibition of COX-2 and
iNOS has
been shown to reduce the accumulation of MDSCs, restore cytotoxic activity of
tumor-
associated T cells, and delay tumor growth (Sinha et al., 2007; Mazzoni et
al., 2002; Zhou et
al., 2007).
Inhibition of the NF-KB and JAK/STAT signaling pathways has been implicated as
a
strategy to inhibit proliferation of cancer epithelial cells and induce their
apoptosis.
Activation of STAT3 and NF-KB has been shown to result in suppression of
apoptosis in
cancer cells, and promotion of proliferation, invasion, and metastasis. Many
of the target
genes involved in these processes have been shown to be transcriptionally
regulated by both
NF-KB and STAT3 (Yu et al., 2007).
In addition to their direct roles in cancer epithelial cells, NF-KB and STAT3
also have
important roles in other cells found within the tumor microenvironment.
Experiments in
animal models have demonstrated that NF-KB is required in both cancer cells
and
hematopoeitic cells to propagate the effects of inflammation on cancer
initiation and
progression (Greten et al., 2004). NF-KB inhibition in cancer and myeloid
cells reduces the
number and size, respectively, of the resultant tumors. Activation of STAT3 in
cancer cells
results in the production of several cytokines (IL-6, IL-10) which suppress
the maturation of
tumor-associated dendritic cells (DC). Furthermore, STAT3 is activated by
these cytokines
in the dendritic cells themselves. Inhibition of STAT3 in mouse models of
cancer restores
DC maturation, promotes antitumor immunity, and inhibits tumor growth
(Kortylewski et al.,
2005).
B. Treatment of Multiple Sclerosis and Other Neurodegenerative Conditions
The compounds and methods of this invention may be used for treating patients
for
multiple sclerosis (MS). MS is known to be an inflammatory condition of the
central nervous
system (Williams et al., 1994; Merrill and Benvenist, 1996; Genain and Nauser,
1997).
Based on several investigations, there is evidence suggesting that
inflammatory, oxidative,
and/or immune mechanisms are involved in the pathogenesis of Alzheimer's
disease (AD),
Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and MS (Bagasra
et al., 1995;
68
CA 02721837 2015-07-30
McGeer and McGeer, 1995; Simonian and Coyle, 1996; Kaltschmidt et al., 1997).
Both
reactive astrocytes and activated microglia have been implicated in causation
of
neurodegenerative disease (NDD) and neuroinflammatory disease (NID); there has
been a
particular emphasis on microglia as cells that synthesize both NO and
prostaglandins as
products of the respective enzymes, iNOS and COX-2. De novo fofination of
these enzymes
may be driven by inflammatory cytokines such as interferon-y or interleukin-1.
In turn,
excessive production of NO may lead to inflammatory cascades and/or oxidative
damage in
cells and tissues of many organs, including neurons and oligodendrocytes of
the nervous
system, with consequent manifestations in AD and MS, and possible PD and ALS
(Coyle and
Puttfarcken, 1993; Beal, 1996; Merrill and Benvenist, 1996; Simonian and
Coyle, 1996;
Vodovotz et al., 1996). Epidemiologic data indicate that chronic use of
NSAID's which
block synthesis of prostaglandins from arachidonate, markedly lower the risk
for
development of AD (McGeer et al., 1996; Stewart et al., 1997). Thus, agents
that block
formation of NO and prostaglandins, may be used in approaches to prevention
and treatment
of NDD. Successful therapeutic candidates for treating such a disease
typically require an
ability to penetrate the blood-brain barrier. See, for example, U.S. Patent
Publication
2009/0060873.
Sec also, for
example, the results presented for compound 404-02 in Examples 4 and 5, below.
C. Neuroinflammation
The compounds and methods of this invention may be used for treating patients
with
neuroinflammation. Neuroinflammation encapsulates the idea that microglial and
astrocytic
responses and actions in the central nervous system have a fundamentally
inflammation-like
character, and that these responses are central to the pathogenesis and
progression of a wide
variety of neurological disorders. This idea originated in the field of
Alzheimer's disease
(Griffin et al., 1989; Rogers et al., 1988), where it has revolutionized our
understanding of
this disease (Akiyama et al., 2000). These ideas have been extended to other
neurodegenerative diseases (Eikelenboom et al., 2002; lshizawa and Dickson,
2001), to
ischemic/toxic diseases (Gehfinann et al., 1995; Touzani et al., 1999), to
tumor biology
(Graeber et al., 2002) and even to normal brain development.
Neuroinflammation incorporates a wide spectrum of complex cellular responses
that
include activation of microglia and astrocytes and induction of cytokines,
chemokines,
complement proteins, acute phase proteins, oxidative injury, and related
molecular processes.
69
CA 02721837 2015-07-30
"f hese events may have detrimental effects on neuronal function, leading to
neuronal injury,
further glial activation, and ultimately neurodegeneration.
D. Treatment of Renal Failure
The compounds and methods of this invention may be used for treating patients
with
renal failure. See U.S. Patent Application Publication No. 2009-0326063.
Another aspect of the present disclosure concerns new methods and
compounds for the treatment and prevention of renal disease. Renal failure,
resulting in
inadequate clearance of metabolic waste products from the blood and abnormal
concentrations of electrolytes in the blood, is a significant medical problem
throughout the
world, especially in developed countries. Diabetes and hypertension are among
the most
important causes of chronic renal failure, also known as chronic kidney
disease (CKD), but it
is also associated with other conditions such as lupus. Acute renal failure
may arise from
exposure to certain drugs (e.g., acetaminophen) or toxic chemicals, or from
ischemia-
reperfusion injury associated with shock or surgical procedures such as
transplantation, and
may result in chronic renal failure. In many patients, renal failure advances
to a stage in
which the patient requires regular dialysis or kidney transplantation to
continue living. Both
of these procedures are highly invasive and associated with significant side
effects and quality
of life issues. Although there are effective treatments for some complications
of renal failure,
such as hyperparathyroidism and hyperphosphatemia, no available treatment has
been shown
to halt or reverse the underlying progression of renal failure. Thus, agents
that can improve
compromised renal function would represent a significant advance in the
treatment of renal
failure.
Inflammation contributes significantly to the pathology of CKD. There is also
a
strong mechanistic link between oxidative stress and renal dysfunction. The NF-
KB signaling
pathway plays an important role in the progression of CKD as NF-KB regulates
the
transcription of MCP-1, a chcmokine that is responsible for the recruitment of
monocytes/macrophages resulting in an inflammatory response that ultimately
injures the
kidney (Wardle, 2001). The Keapl/Nrf2/ARE pathway controls the transcription
of several
genes encoding antioxidant enzymes, including heme oxygenase-1 (H0-1).
Ablation of the
Nrf2 gene in female mice results in the development of lupus-like glomerular
nephritis (Yoh
et al., 2001). Furthermore, several studies have demonstrated that HO-1
expression is
CA 02721837 2015-07-30
induced in response to renal damage and inflammation and that this enzyme and
its products
¨ bilirubin and carbon monoxide ¨ play a protective role in the kidney (Nath
et al., 2006).
The glomerulus and the surrounding Bowman's capsule constitute the basic
functional
unit of the kidney. Glomerular filtration rate (GFR) is the standard measure
of renal function.
Creatinine clearance is commonly used to measure GFR. However, the level of
serum
creatinine is commonly used as a surrogate measure of creatinine clearance.
For instance,
excessive levels of serum creatinine are generally accepted to indicate
inadequate renal
function and reductions in serum creatinine over time are accepted as an
indication of
improved renal function. Normal levels of creatinine in the blood are
approximately 0.6 to
E2 milligrams (mg) per deciliter (dl) in adult males and 0.5 to 1.1 milligrams
per deciliter in
adult females.
Acute kidney injury (AKI) can occur following ischemia-rcperfusion, treatment
with
certain pharmacological agents such as cisplatin and rapamycin, and
intravenous injection of
radiocontrast media used in medical imaging. As in CKD, inflammation and
oxidative stress
contribute to the pathology of AK1. The molecular mechanisms underlying
radiocontrast-
induced nephropathy (RCN) are not well understood; however, it is likely that
a combination
of events including prolonged vasoconstriction, impaired kidney
autoregulation, and direct
toxicity of the contrast media all contribute to renal failure (Tumlin et al.,
2006).
Vasoconstriction results in decreased renal blood flow and causes ischemia-
reperfusion and
the production of reactive oxygen species. HO-1 is strongly induced under
these conditions
and has been demonstrated to prevent ischemia-rcperfusion injury in several
different organs,
including the kidney (Nath et al., 2006). Specifically, induction of HO-1 has
been shown to
be protective in a rat model of RCN (Goodman et al., 2007). Reperfusion also
induces an
inflammatory response, in part though activation of NF-KB signaling (Nichols,
2004).
Targeting NF-KB has been proposed as a therapeutic strategy to prevent organ
damage
(Zingarelli et al., 2003).
E. Cardiovascular Disease
The compounds and methods of this invention may be used for treating patients
with
cardiovascular disease. See U.S. Patent Application Publication No. 2009-
0326063.
Cardiovascular (CV) disease is among the most important
cauSes of mortality worldwide, and is the leading cause of death in many
developed nations.
The etiology of CV disease is complex, but the majority of causes are related
to inadequate or
71
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
completely disrupted supply of blood to a critical organ or tissue. Frequently
such a
condition arises from the rupture of one or more atherosclerotic plaques,
which leads to the
formation of a thrombus that blocks blood flow in a critical vessel. Such
thrombosis is the
principal cause of heart attacks, in which one or more of the coronary
arteries is blocked and
blood flow to the heart itself is disrupted. The resulting ischemia is highly
damaging to
cardiac tissue, both from lack of oxygen during the ischemic event and from
excessive
formation of free radicals after blood flow is restored (a phenomenon known as
ischemia-
reperfusion injury). Similar damage occurs in the brain during a thrombotic
stroke, when a
cerebral artery or other major vessel is blocked by thrombosis. Hemorrhagic
strokes, in
contrast, involve rupture of a blood vessel and bleeding into the surrounding
brain tissue.
This creates oxidative stress in the immediate area of the hemorrhage, due to
the presence of
large amounts of free heme and other reactive species, and ischemia in other
parts of the brain
due to compromised blood flow.
Subarachnoid hemorrhage, which is frequently
accompanied by cerebral vasospasm, also causes ischemia/reperfusion injury in
the brain.
Alternatively, atherosclerosis may be so extensive in critical blood vessels
that
stenosis (narrowing of the arteries) develops and blood flow to critical
organs (including the
heart) is chronically insufficient. Such chronic ischemia can lead to end-
organ damage of
many kinds, including the cardiac hypertrophy associated with congestive heart
failure.
Atherosclerosis, the underlying defect leading to many forms of cardiovascular
disease, occurs when a physical defect or injury to the lining (endothelium)
of an artery
triggers an inflammatory response involving the proliferation of vascular
smooth muscle cells
and the infiltration of leukocytes into the affected area. Ultimately, a
complicated lesion
known as an atherosclerotic plaque may form, composed of the above-mentioned
cells
combined with deposits of cholesterol-bearing lipoproteins and other materials
(e.g., Hansson
et al., 2006).
Pharmaceutical treatments for cardiovascular disease include preventive
treatments,
such as the use of drugs intended to lower blood pressure or circulating
levels of cholesterol
and lipoproteins, as well as treatments designed to reduce the adherent
tendencies of platelets
and other blood cells (thereby reducing the rate of plaque progression and the
risk of
thrombus formation). More recently, drugs such as streptokinase and tissue
plasminogen
activator have been introduced and are used to dissolve the thrombus and
restore blood flow.
Surgical treatments include coronary artery bypass grafting to create an
alternative blood
supply, balloon angioplasty to compress plaque tissue and increase the
diameter of the arterial
72
CA 02721837 2015-07-30
lumen, and carotid endarterectomy to remove plaque tissue in the carotid
artery. Such
treatments, especially balloon angioplasty, may be accompanied by the use of
stents,
expandable mesh tubes designed to support the artery walls in the affected
area and keep the
vessel open. Recently, the use of drug-eluting stents has become common in
order to prevent
post-surgical restenosis (renarrowing of the artery) in the affected area.
These devices are
wire stents coatcd with a biocompatible polymer matrix containing a drug that
inhibits cell
proliferation (e.g., paclitaxel or rapamycin). The polymer allows a slow,
localized release of
the drug in the affected area with minimal exposure of non-target tissues.
Despite the
significant benefits offered by such treatments, mortality from cardiovascular
disease remains
high and significant unmet needs in the treatment of cardiovascular disease
remain.
As noted above, induction of 1-10-1 has been shown to be beneficial in a
variety of
models of cardiovascular disease, and low levels of H0-1 expression have been
clinically
correlated with elevated risk of CV disease. Compounds of thc invention,
therefore, may be
used in. treating or preventing a variety of cardiovascular disorders
including but not limited
to atherosclerosis, hypertension, myocardial infarction, chronic heart
failure, stroke,
subarachnoid hemorrhage, and restenosis.
F. Diabetes
The compounds and methods of this invention may be used for treating patients
with
diabctcs. See U.S. Patent Application Publication No. 2009-0326063.
Diabetes is a complex disease characterized by the body's failure to regulate
circulating levels of glucose. This failure may result from a lack of insulin,
a peptide
hormone that regulates both the production and absorption of glucose in
various tissues.
Deficient insulin compromises the ability of muscle, fat, and other tissues to
absorb glucose
properly, leading to hyperglycemia (abnormally high levels of glucose in the
blood). Most
commonly, such insulin deficiency results from inadequate production in the
islet cells of the
pancreas. In the majority of cases this arises from autoimmune destruction of
these cells, a
condition known as type 1 or juvenile-onset diabetes, but may also bc due to
physical trauma
or some other cause.
Diabetes may also arise when muscle and fat cells become less responsive to
insulin
and do not absorb glucose properly, resulting in hyperglycemia. This
phenomenon is known
as insulin resistance, and the resulting condition is known as Type 2
diabetes. Type 2
diabetes, the most common type, is highly associated with obesity and
hypertension. Obesity
73
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
is associated with an inflammatory state of adipose tissue that is thought to
play a major role
in the development of insulin resistance (e.g., Hotamisligil, 2006; Guilherme
et al., 2008).
Diabetes is associated with damage to many tissues, largely because
hyperglycemia
(and hypoglycemia, which can result from excessive or poorly timed doses of
insulin) is a
significant source of oxidative stress. Chronic kidney failure, retinopathy,
peripheral
neuropathy, peripheral vasculitis, and the development of dermal ulcers that
heal slowly or
not at all are among the common complications of diabetes. Because of their
ability to
protect against oxidative stress, particularly by the induction of HO-1
expression, compounds
of the invention may be used in treatments for many complications of diabetes.
As noted
above (Cai et al., 2005), chronic inflammation and oxidative stress in the
liver are suspected
to be primary contributing factors in the development of Type 2 diabetes.
Furthermore,
PPARy agonists such as thiazolidinediones are capable of reducing insulin
resistance and are
known to be effective treatments for Type 2 diabetes.
The effect of treatment of diabetes may be evaluated as follows. Both the
biological
efficacy of the treatment modality as well as the clinical efficacy are
evaluated, if possible.
For example, because the disease manifests itself by increased blood sugar,
the biological
efficacy of the treatment therefore can be evaluated, for example, by
observation of return of
the evaluated blood glucose towards normal. Measurement of glycosylated
hemoglobin, also
called Al c or HbAl c, is another commonly used parameter of blood glucose
control.
Measuring a clinical endpoint which can give an indication of b-cell
regeneration after, for
example, a six-month period of time, can give an indication of the clinical
efficacy of the
treatment regimen.
G. Rheumatoid Arthritis
The compounds and methods of this invention may be used for treating patients
with
RA. Typically the first signs of rheumatoid arthritis (RA) appear in the
synovial lining layer,
with proliferation of synovial fibroblasts and their attachment to the
articular surface at the
joint margin (Lipsky, 1998). Subsequently, macrophages, T cells and other
inflammatory cells
are recruited into the joint, where they produce a number of mediators,
including the
cytokines interleukin-1 (IL-1), which contributes to the chronic sequelae
leading to bone and
cartilage destruction, and tumour necrosis factor (TNF-a), which plays a role
in inflammation
(Dinarello, 1998; Arend and Dayer, 1995; van den Berg, 2001). The
concentration of IL-1 in
plasma is significantly higher in patients with RA than in healthy individuals
and, notably,
74
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
plasma IL-1 levels correlate with RA disease activity (Eastgate et al., 1988).
Moreover,
synovial fluid levels of IL-1 are correlated with various radiographic and
histologic features
of RA (Kahle et al., 1992; Rooney et al., 1990).
In normal joints, the effects of these and other proinflammatory cytokines are
balanced by a variety of anti-inflammatory cytokines and regulatory factors
(Burger and
Dayer, 1995). The significance of this cytokine balance is illustrated in
juvenile RA patients,
who have cyclical increases in fever throughout the day (Prieur et al., 1987).
After each peak
in fever, a factor that blocks the effects of IL-1 is found in serum and
urine. This factor has
been isolated, cloned and identified as IL-1 receptor antagonist (IL- lra), a
member of the IL-1
gene family (Hannum et al., 1990). IL- lra, as its name indicates, is a
natural receptor
antagonist that competes with IL-1 for binding to type I IL-1 receptors and,
as a result, blocks
the effects of IL-1 (Arend et al., 1998). A 10- to 100-fold excess of IL- lra
may be needed to
block IL-1 effectively; however, synovial cells isolated from patients with RA
do not appear
to produce enough IL- lra to counteract the effects of IL-1 (Firestein et al.,
1994; Fujikawa et
al., 1995).
H. Psoriatic Arthritis
The compounds and methods of this invention may be used for treating patients
with
psoriatic arthritis. Psoriasis is an inflammatory and proliferative skin
disorder with a
prevalence of 1.5-3%. Approximately 20% of patients with psoriasis develop a
characteristic
form of arthritis that has several patterns (Gladman, 1992; Jones et al.,
1994; Gladman et al.,
1995). Some individuals present with joint symptoms first but in the majority,
skin psoriasis
presents first. About one-third of patients have simultaneous exacerbations of
their skin and
joint disease (Gladman et al., 1987) and there is a topographic relationship
between nail and
distal interphalangeal joint disease (Jones et al., 1994; Wright, 1956).
Although the
inflammatory processes which link skin, nail and joint disease remain elusive,
an immune-
mediated pathology is implicated.
Psoriatic arthritis (PsA) is a chronic inflammatory arthropathy characterized
by the
association of arthritis and psoriasis and was recognized as a clinical entity
distinct from
rheumatoid arthritis (RA) in 1964 (Blumberg et al., 1964). Subsequent studies
have revealed
that PsA shares a number of genetic, pathogenic and clinical features with
other
spondyloarthropathies (SpAs), a group of diseases that comprise ankylosing
spondylitis,
reactive arthritis and enteropathic arthritis (Wright, 1979). The notion that
PsA belongs to the
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
SpA group has recently gained further support from imaging studies
demonstrating
widespread enthesitis in the, including PsA but not RA (McGonagle et al.,
1999; McGonagle
et al., 1998). More specifically, enthesitis has been postulated to be one of
the earliest events
occurring in the SpAs, leading to bone remodeling and ankylosis in the spine,
as well as to
articular synovitis when the inflamed entheses are close to peripheral joints.
However, the
link between enthesitis and the clinical manifestations in PsA remains largely
unclear, as PsA
can present with fairly heterogeneous patterns of joint involvement with
variable degrees of
severity (Marsal et al., 1999; Salvarani et al., 1998). Thus, other factors
must be posited to
account for the multifarious features of PsA, only a few of which (such as the
expression of
the HLA-B27 molecule, which is strongly associated with axial disease) have
been identified.
As a consequence, it remains difficult to map the disease manifestations to
specific
pathogenic mechanisms, which means that the treatment of this condition
remains largely
empirical.
Family studies have suggested a genetic contribution to the development of PsA
(Moll
and Wright, 1973). Other chronic inflammatory forms of arthritis, such as
ankylosing
spondylitis and rheumatoid arthritis, are thought to have a complex genetic
basis. However,
the genetic component of PsA has been difficult to assess for several reasons.
There is strong
evidence for a genetic predisposition to psoriasis alone that may mask the
genetic factors that
are important for the development of PsA. Although most would accept PsA as a
distinct
disease entity, at times there is a phenotypic overlap with rheumatoid
arthritis and ankylosing
spondylitis. Also, PsA itself is not a homogeneous condition and various
subgroups have been
proposed.
Increased amounts of TNF-a have been reported in both psoriatic skin (Ettehadi
et al.,
1994) and synovial fluid (Partsch et al., 1997). Recent trials have shown a
positive benefit of
anti-TNF treatment in both PsA (Mease et al., 2000) and ankylosing spondylitis
(Brandt et al.,
2000).
I. Reactive Arthritis
The compounds and methods of this invention may be used for treating patients
with
reactive arthritis. In reactive arthritis (ReA) the mechanism of joint damage
is unclear, but it
is likely that cytokines play critical roles. A more prevalent Thl profile
high levels of
interferon gamma (IFN-y) and low levels of interleukin 4 (IL-4) has been
reported (Lahesmaa
et al., 1992; Schlaak et al., 1992; Simon et al., 1993; Schlaak et al., 1996;
Kotake et al.,
76
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
1999; Ribbens et al., 2000), but several studies have shown relative
predominance of IL-4 and
IL-10 and relative lack of IFN-y and tumour necrosis factor alpha (TNF-a) in
the synovial
membrane (Simon et al., 1994; Yin et al., 1999) and fluid (SF) (Yin et al.,
1999; Yin et al.,
1997) of reactive arthritis patients compared with rheumatoid arthritis (RA)
patients. A lower
level of TNF-a secretion in reactive arthritis than in RA patients has also
been reported after
ex vivo stimulation of peripheral blood mononuclear cells (PBMC) (Braun et
al., 1999).
It has been argued that clearance of reactive arthritis-associated bacteria
requires the
production of appropriate levels of IFN-y and TNF-a, while IL-10 acts by
suppressing these
responses (Autenrieth et al., 1994; Sieper and Braun, 1995). IL-10 is a
regulatory cytokine
that inhibits the synthesis of IL-12 and TNF-y by activated macrophages (de
Waal et al.,
1991; Hart et al., 1995; Chomarat et al., 1995) and of IFN-y by T cells
(Macatonia et al.,
1993).
J. Enteropathic Arthritis
The compounds and methods of this invention may be used for treating patients
with
enteropathic arthritis. Typically enteropathic arthritis (EA) occurs in
combination with
inflammatory bowel diseases (IBD) such as Crohn's disease or ulcerative
colitis. It also can
affect the spine and sacroiliac joints. Enteropathic arthritis involves the
peripheral joints,
usually in the lower extremities such as the knees or ankles. It commonly
involves only a few
or a limited number of joints and may closely follow the bowel condition. This
occurs in
approximately 11% of patients with ulcerative colitis and 21% of those with
Crohn's disease.
The synovitis is generally self-limited and non-deforming.
Enteropathic arthropathies comprise a collection of rheumatologic conditions
that
share a link to GI pathology. These conditions include reactive (i.e.,
infection-related)
arthritis due to bacteria (e.g., Shigella, Salmonella, Campylobacter, Yersinia
species,
Clostridium difficile), parasites (e.g., Strongyloides stercoralis, Taenia
saginata, Giardia
lamblia, Ascaris lumbricoides, Cryptosporidium species), and
spondyloarthropathies
associated with inflammatory bowel disease (IBD). Other conditions and
disorders include
intestinal bypass (jejunoileal), arthritis, celiac disease, Whipple disease,
and collagenous
colitis.
77
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
K. Juvenile Rheumatoid Arthritis
The compounds and methods of this invention may be used for treating patients
with
JRA. Juvenile rheumatoid arthritis (JRA), a term for the most prevalent form
of arthritis in
children, is applied to a family of illnesses characterized by chronic
inflammation and
hypertrophy of the synovial membranes. The term overlaps, but is not
completely
synonymous, with the family of illnesses referred to as juvenile chronic
arthritis and/or
juvenile idiopathic arthritis in Europe.
Both innate and adaptive immune systems use multiple cell types, a vast array
of cell
surface and secreted proteins, and interconnected networks of positive and
negative feedback
(Lo et al., 1999). Furthermore, while separable in thought, the innate and
adaptive wings of
the immune system are functionally intersected (Fearon and Locksley, 1996),
and pathologic
events occurring at these intersecting points are likely to be highly relevant
to our
understanding of pathogenesis of adult and childhood forms of chronic
arthritis (Warrington,
et al., 2001).
Polyarticular JRA is a distinct clinical subtype characterized by inflammation
and
synovial proliferation in multiple joints (four or more), including the small
joints of the hands
(Jarvis, 2002). This subtype of JRA may be severe, because of both its
multiple joint
involvement and its capacity to progress rapidly over time. Although
clinically distinct,
polyarticular JRA is not homogeneous, and patients vary in disease
manifestations, age of
onset, prognosis, and therapeutic response. These differences very likely
reflect a spectrum of
variation in the nature of the immune and inflammatory attack that can occur
in this disease
(Jarvis, 1998).
L. Early Inflammatory Arthritis
The compounds and methods of this invention may be used for treating patients
with
early inflammatory arthritis. The clinical presentation of different
inflammatory arthropathies
is similar early in the course of disease. As a result, it is often difficult
to distinguish patients
who are at risk of developing the severe and persistent synovitis that leads
to erosive joint
damage from those whose arthritis is more self-limited. Such distinction is
critical in order to
target therapy appropriately, treating aggressively those with erosive disease
and avoiding
unnecessary toxicity in patients with more self-limited disease. Current
clinical criteria for
diagnosing erosive arthropathies such as rheumatoid arthritis (RA) are less
effective in early
disease and traditional markers of disease activity such as joint counts and
acute phase
78
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
response do not adequately identify patients likely to have poor outcomes
(Harrison et al.,
1998). Parameters reflective of the pathologic events occurring in the
synovium are most
likely to be of significant prognostic value.
Recent efforts to identify predictors of poor outcome in early inflammatory
arthritis
have identified the presence of RA specific autoantibodies, in particular
antibodies towards
citrullinated peptides, to be associated with erosive and persistent disease
in early
inflammatory arthritis cohorts. On the basis of this, a cyclical citrullinated
peptide (CCP) has
been developed to assist in the identification of anti-CCP antibodies in
patient sera. Using
this approach, the presence of anti-CCP antibodies has been shown to be
specific and
sensitive for RA, can distinguish RA from other arthropathies, and can
potentially predict
persistent, erosive synovitis before these outcomes become clinically
manifest. Importantly,
anti-CCP antibodies are often detectable in sera many years prior to clinical
symptoms
suggesting that they may be reflective of subclinical immune events (Nielen et
al., 2004;
Rantapaa-Dahlqvist et al., 2003).
M. Ankylosing Spondylitis
The compounds and methods of this invention may be used for treating patients
with
ankylosing spondylitis. AS is a disease subset within a broader disease
classification of
spondyloarthropathy. Patients affected with the various subsets of
spondyloarthropathy have
disease etiologies that are often very different, ranging from bacterial
infections to
inheritance. Yet, in all subgroups, the end result of the disease process is
axial arthritis.
Despite the early clinically differences seen in the various patient
populations, many of them
end up nearly identical after a disease course of ten-to-twenty years. Recent
studies suggest
the mean time to clinical diagnosis of ankylosing spondylitis from disease
onset of disease is
7.5 years (Khan, 1998). These same studies suggest that the
spondyloarthropathies may have
prevalence close to that of rheumatoid arthritis (Feldtkeller et al., 2003;
Doran et al., 2003).
AS is a chronic systemic inflammatory rheumatic disorder of the axial skeleton
with
or without extraskeletal manifestations. Sacroiliac joints and the spine are
primarily affected,
but hip and shoulder joints, and less commonly peripheral joints or certain
extra-articular
structures such as the eye, vasculature, nervous system, and gastrointestinal
system may also
be involved. Its etiology is not yet fully understood (Wordsworth, 1995; Calin
and Taurog,
1998). It is strongly associated with the major histocompatibility class I
(MHC I) HLA-B27
allele (Calin and Taurog, 1998). AS affects individuals in the prime of their
life and is feared
79
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
because of its potential to cause chronic pain and irreversible damage of
tendons, ligaments,
joints, and bones (Brewerton et al., 1973a; Brewerton et al., 1973b;
Schlosstein et al., 1973).
AS may occur alone or in association with another form of spondyloarthropathy
such as
reactive arthritis, psoriasis, psoriatic arthritis, enthesitis, ulcerative
colitis, irritable bowel
disease, or Crohn's disease, in which case it is classified as secondary AS.
Typically, the affected sites include the discovertebral, apophyseal,
costovertebral,
and costotransverse joints of the spine, and the paravertebral ligamentous
structures.
Inflammation of the entheses, which are sites of musculotendinous and
ligamentous
attachment to bones, is also prominent in this disease (Calin and Taurog,
1998). The site of
enthesitis is known to be infiltrated by plasma cells, lymphocytes, and
polymorphonuclear
cells. The inflammatory process frequently results in gradual fibrous and bony
ankylosis,
(Ball, 1971; Khan, 1990).
Delayed diagnosis is common because symptoms are often attributed to more
common back problems. A dramatic loss of flexibility in the lumbar spine is an
early sign of
AS. Other common symptoms include chronic pain and stiffness in the lower back
which
usually starts where the lower spine is joined to the pelvis, or hip. Although
most symptoms
begin in the lumbar and sacroiliac areas, they may involve the neck and upper
back as well.
Arthritis may also occur in the shoulder, hips and feet. Some patients have
eye inflammation,
and more severe cases must be observed for heart valve involvement.
The most frequent presentation is back pain, but disease can begin atypically
in
peripheral joints, especially in children and women, and rarely with acute
iritis (anterior
uveitis). Additional early symptoms and signs are diminished chest expansion
from diffuse
costovertebral involvement, low-grade fever, fatigue, anorexia, weight loss,
and anemia.
Recurrent back pain - often nocturnal and of varying intensity - is an
eventual complaint, as is
morning stiffness typically relieved by activity. A flexed or bent-over
posture eases back pain
and paraspinal muscle spasm; thus, some degree of kyphosis is common in
untreated patients.
Systemic manifestations occur in 1/3 of patients. Recurrent, usually self-
limited, acute
iritis (anterior uveitis) rarely is protracted and severe enough to impair
vision. Neurologic
signs can occasionally result from compression radiculitis or sciatica,
vertebral fracture or
subluxation, and cauda equina syndrome (which consists of impotence, nocturnal
urinary
incontinence, diminished bladder and rectal sensation, and absence of ankle
jerks).
Cardiovascular manifestations can include aortic insufficiency, angina,
pericarditis, and ECG
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
conduction abnormalities. A rare pulmonary finding is upper lobe fibrosis,
occasionally with
cavitation that may be mistaken for TB and can be complicated by infection
with Aspergillus.
AS is characterized by mild or moderate flares of active spondylitis
alternating with
periods of almost or totally inactive inflammation. Proper treatment in most
patients results in
minimal or no disability and in full, productive lives despite back stiffness.
Occasionally, the
course is severe and progressive, resulting in pronounced incapacitating
deformities. The
prognosis is bleak for patients with refractory iritis and for the rare
patient with secondary
amyloidosis.
N. Ulcerative Colitis
The compounds and methods of this invention may be used for treating patients
with
ulcerative colitis. Ulcerative colitis is a disease that causes inflammation
and sores, called
ulcers, in the lining of the large intestine. The inflammation usually occurs
in the rectum and
lower part of the colon, but it may affect the entire colon. Ulcerative
colitis rarely affects the
small intestine except for the end section, called the terminal ileum.
Ulcerative colitis may
also be called colitis or proctitis. The inflammation makes the colon empty
frequently,
causing diarrhea. Ulcers form in places where the inflammation has killed the
cells lining the
colon; the ulcers bleed and produce pus.
Ulcerative colitis is an inflammatory bowel disease (IBD), the general name
for
diseases that cause inflammation in the small intestine and colon. Ulcerative
colitis can be
difficult to diagnose because its symptoms are similar to other intestinal
disorders and to
another type of IBD, Crohn's disease. Crohn's disease differs from ulcerative
colitis because
it causes inflammation deeper within the intestinal wall. Also, Crohn's
disease usually occurs
in the small intestine, although it can also occur in the mouth, esophagus,
stomach,
duodenum, large intestine, appendix, and anus.
Ulcerative colitis may occur in people of any age, but most often it starts
between
ages 15 and 30, or less frequently between ages 50 and 70. Children and
adolescents
sometimes develop the disease. Ulcerative colitis affects men and women
equally and appears
to run in some families. Theories about what causes ulcerative colitis abound,
but none have
been proven. The most popular theory is that the body's immune system reacts
to a virus or a
bacterium by causing ongoing inflammation in the intestinal wall. People with
ulcerative
colitis have abnormalities of the immune system, but doctors do not know
whether these
abnormalities are a cause or a result of the disease. Ulcerative colitis is
not caused by
81
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
emotional distress or sensitivity to certain foods or food products, but these
factors may
trigger symptoms in some people.
The most common symptoms of ulcerative colitis are abdominal pain and bloody
diarrhea. Patients also may experience fatigue, weight loss, loss of appetite,
rectal bleeding,
and loss of body fluids and nutrients. About half of patients have mild
symptoms. Others
suffer frequent fever, bloody diarrhea, nausea, and severe abdominal cramps.
Ulcerative
colitis may also cause problems such as arthritis, inflammation of the eye,
liver disease
(hepatitis, cirrhosis, and primary sclerosing cholangitis), osteoporosis, skin
rashes, and
anemia. No one knows for sure why problems occur outside the colon. Scientists
think these
complications may occur when the immune system triggers inflammation in other
parts of the
body. Some of these problems go away when the colitis is treated.
A thorough physical exam and a series of tests may be required to diagnose
ulcerative
colitis. Blood tests may be done to check for anemia, which could indicate
bleeding in the
colon or rectum. Blood tests may also uncover a high white blood cell count,
which is a sign
of inflammation somewhere in the body. By testing a stool sample, the doctor
can detect
bleeding or infection in the colon or rectum. The doctor may do a colonoscopy
or
sigmoidoscopy. For either test, the doctor inserts an endoscope - a long,
flexible, lighted tube
connected to a computer and TV monitor - into the anus to see the inside of
the colon and
rectum. The doctor will be able to see any inflammation, bleeding, or ulcers
on the colon
wall. During the exam, the doctor may do a biopsy, which involves taking a
sample of tissue
from the lining of the colon to view with a microscope. A barium enema x ray
of the colon
may also be required. This procedure involves filling the colon with barium, a
chalky white
solution. The barium shows up white on x-ray film, allowing the doctor a clear
view of the
colon, including any ulcers or other abnormalities that might be there.
Treatment for ulcerative colitis depends on the seriousness of the disease.
Most people
are treated with medication. In severe cases, a patient may need surgery to
remove the
diseased colon. Surgery is the only cure for ulcerative colitis. Some people
whose symptoms
are triggered by certain foods are able to control the symptoms by avoiding
foods that upset
their intestines, like highly seasoned foods, raw fruits and vegetables, or
milk sugar (lactose).
Each person may experience ulcerative colitis differently, so treatment is
adjusted for each
individual. Emotional and psychological support is important. Some people have
remissions
- periods when the symptoms go away - that last for months or even years.
However, most
patients' symptoms eventually return. This changing pattern of the disease
means one cannot
82
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
always tell when a treatment has helped. Some people with ulcerative colitis
may need
medical care for some time, with regular doctor visits to monitor the
condition.
O. Crohn's Disease
The compounds and methods of this invention may be used for treating patients
with
Crohn's disease. Another disorder for which immunosuppression has been tried
is Crohn's
disease. Crohn's disease symptoms include intestinal inflammation and the
development of
intestinal stenosis and fistulas; neuropathy often accompanies these symptoms.
Anti-
inflammatory drugs, such as 5-aminosalicylates (e.g., mesalamine) or
corticosteroids, are
typically prescribed, but are not always effective (reviewed in Botoman et
al., 1998).
Immunosuppression with cyclosporine is sometimes beneficial for patients
resistant to or
intolerant of corticosteroids (Brynskov et al., 1989).
Efforts to develop diagnostic and treatment tools against Crohn's disease have
focused
on the central role of cytokines (Schreiber, 1998; van Hogezand and Verspaget,
1998).
Cytokines are small secreted proteins or factors (5 to 20 kD) that have
specific effects on cell-
to-cell interactions, intercellular communication, or the behavior of other
cells. Cytokines are
produced by lymphocytes, especially TH1 and TH2 lymphocytes, monocytes,
intestinal
macrophages, granulocytes, epithelial cells, and fibroblasts (reviewed in
Rogler and. Andus,
1998; Galley and Webster, 1996). Some cytokines are pro-inflammatory (e.g.,
TNF-a, IL-1(a
and (3), IL-6, IL-8, IL-12, or leukemia inhibitory factor [LIF]); others are
anti-inflammatory
(e.g., IL-1 receptor antagonist, IL-4, IL-10, IL-11, and TGF-I3). However,
there may be
overlap and functional redundancy in their effects under certain inflammatory
conditions.
In active cases of Crohn's disease, elevated concentrations of TNF-a and IL-6
are
secreted into the blood circulation, and TNF-a, IL-1, IL-6, and IL-8 are
produced in excess
locally by mucosal cells (id.; Funakoshi et al., 1998). These cytokines can
have far-ranging
effects on physiological systems including bone development, hematopoiesis,
and liver,
thyroid, and neuropsychiatric function. Also, an imbalance of the IL-113/IL-
lra ratio, in favor
of pro-inflammatory IL-113, has been observed in patients with Crohn's disease
(Rogler and
Andus, 1998; Saiki et al., 1998; Dionne et al., 1998; but see Kuboyama, 1998).
One study
suggested that cytokine profiles in stool samples could be a useful diagnostic
tool for Crohn's
disease (Saiki et al., 1998).
Treatments that have been proposed for Crohn's disease include the use of
various
cytokine antagonists (e.g., IL- lra), inhibitors (e.g., of IL-1I3 converting
enzyme and
83
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
antioxidants) and anti-cytokine antibodies (Rogler and Andus, 1998; van
Hogezand and
Verspaget, 1998; Reimund et al., 1998; Lugering et al., 1998; McAlindon et
al., 1998). In
particular, monoclonal antibodies against TNF-a have been tried with some
success in the
treatment of Crohn's disease (Targan et al., 1997; Stack et al., 1997; van
Dullemen et al.,
1995). These compounds may be used in combination therapy with compounds of
the present
disclosure.
Another approach to the treatment of Crohn's disease has focused on at least
partially
eradicating the bacterial community that may be triggering the inflammatory
response and
replacing it with a non-pathogenic community. For example, U.S. Patent
5,599,795 discloses
a method for the prevention and treatment of Crohn's disease in human
patients. Their
method was directed to sterilizing the intestinal tract with at least one
antibiotic and at least
one anti-fungal agent to kill off the existing flora and replacing them with
different, select,
well-characterized bacteria taken from normal humans. Borody taught a method
of treating
Crohn's disease by at least partial removal of the existing intestinal
microflora by lavage and
replacement with a new bacterial community introduced by fecal inoculum from a
disease-
screened human donor or by a composition comprising Bacteroides and
Escherichia coli
species. (U.S. Patent 5,443,826).
P. Systemic Lupus Erythematosus
The compounds and methods of this invention may be used for treating patients
with
SLE. There has also been no known cause for autoimmune diseases such as
systemic lupus
erythematosus. Systemic lupus erythematosus (SLE) is an autoimmune rheumatic
disease
characterized by deposition in tissues of autoantibodies and immune complexes
leading to
tissue injury (Kotzin, 1996). In contrast to autoimmune diseases such as MS
and type 1
diabetes mellitus, SLE potentially involves multiple organ systems directly,
and its clinical
manifestations are diverse and variable (reviewed by Kotzin and O'Dell, 1995).
For example,
some patients may demonstrate primarily skin rash and joint pain, show
spontaneous
remissions, and require little medication. At the other end of the spectrum
are patients who
demonstrate severe and progressive kidney involvement that requires therapy
with high doses
of steroids and cytotoxic drugs such as cyclophosphamide (Kotzin, 1996).
The serological hallmark of SLE, and the primary diagnostic test available, is
elevated
serum levels of IgG antibodies to constituents of the cell nucleus, such as
double-stranded
DNA (dsDNA), single-stranded DNA (ss-DNA), and chromatin. Among these
84
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
autoantibodies, IgG anti-dsDNA antibodies play a major role in the development
of lupus
glomerulonephritis (G N) (Hahn and Tsao, 1993; Ohnishi et al., 1994).
Glomerulonephritis is
a serious condition in which the capillary walls of the kidney's blood
purifying glomeruli
become thickened by accretions on the epithelial side of glomerular basement
membranes.
The disease is often chronic and progressive and may lead to eventual renal
failure.
Q. Irritable Bowel Syndrome
The compounds and methods of this invention may be used for treating patients
with
Irritable bowel syndrome (IBS). IBS is a functional disorder characterized by
abdominal pain
and altered bowel habits. This syndrome may begin in young adulthood and can
be
associated with significant disability. This syndrome is not a homogeneous
disorder. Rather,
subtypes of IBS have been described on the basis of the predominant symptom--
diarrhea,
constipation, or pain. In the absence of "alarm" symptoms, such as fever,
weight loss, and
gastrointestinal bleeding, a limited workup is needed. Once a diagnosis of IBS
is made, an
integrated treatment approach can effectively reduce the severity of symptoms.
IBS is a
common disorder, although its prevalence rates have varied. In general, IBS
affects about
15% of US adults and occurs about three times more often in women than in men
(Jailwala et
al., 2000).
IBS accounts for between 2.4 million and 3.5 million visits to physicians each
year. It
not only is the most common condition seen by gastroenterologists but also is
one of the most
common gastrointestinal conditions seen by primary care physicians (Everhart
et al., 1991;
Sandler, 1990).
IBS is also a costly disorder. Compared with persons who do not have bowel
symptoms, persons with IBS miss three times as many workdays and are more
likely to report
being too sick to work (Drossman et al., 1993; Drossman et al., 1997).
Moreover, those with
IBS incur hundreds of dollars more in medical charges than persons without
bowel disorders
(Talley et al., 1995).
No specific abnormality accounts for the exacerbations and remissions of
abdominal
pain and altered bowel habits experienced by patients with IBS. The evolving
theory of IBS
suggests dysregulation at multiple levels of the brain-gut axis. Dysmotility,
visceral
hypersensitivity, abnormal modulation of the central nervous system (CNS), and
infection
have all been implicated. In addition, psychosocial factors play an important
modifying role.
Abnormal intestinal motility has long been considered a factor in the
pathogenesis of IBS.
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
Transit time through the small intestine after a meal has been shown to be
shorter in patients
with diarrhea-predominant IBS than in patients who have the constipation-
predominant or
pain-predominant subtype (Cann et al., 1983).
In studies of the small intestine during fasting, the presence of both
discrete, clustered
contractions and prolonged, propagated contractions has been reported in
patients with IBS
(Kellow and Phillips, 1987). They also experience pain with irregular
contractions more often
than healthy persons (Kellow and Phillips, 1987; Horwitz and Fisher, 2001)
These motility findings do not account for the entire symptom complex in
patients
with IBS; in fact, most of these patients do not have demonstrable
abnormalities (Rothstein,
2000). Patients with IBS have increased sensitivity to visceral pain. Studies
involving
balloon distention of the rectosigmoid colon have shown that patients with IBS
experience
pain and bloating at pressures and volumes much lower than control subjects
(Whitehead et
al., 1990). These patients maintain normal perception of somatic stimuli.
Multiple theories have been proposed to explain this phenomenon. For example,
receptors in the viscera may have increased sensitivity in response to
distention or
intraluminal contents. Neurons in the dorsal horn of the spinal cord may have
increased
excitability. In addition, alteration in CNS processing of sensations may be
involved
(Drossman et al., 1997). Functional magnetic resonance imaging studies have
recently shown
that compared with control subjects, patients with IBS have increased
activation of the
anterior cingulate cortex, an important pain center, in response to a painful
rectal stimulus
(Mertz et al., 2000).
Increasingly, evidence suggests a relationship between infectious enteritis
and
subsequent development of IBS. Inflammatory cytokines may play a role. In a
survey of
patients with a history of confirmed bacterial gastroenteritis (Neal et al.,
1997), 25% reported
persistent alteration of bowel habits. Persistence of symptoms may be due to
psychological
stress at the time of acute infection (Gwee et al., 1999).
Recent data suggest that bacterial overgrowth in the small intestine may have
a role in
IBS symptoms. In one study (Pimentel et al., 2000), 157 (78%) of 202 IBS
patients referred
for hydrogen breath testing had test findings that were positive for bacterial
overgrowth. Of
the 47 subjects who had follow-up testing, 25 (53%) reported improvement in
symptoms (i.e.,
abdominal pain and diarrhea) with antibiotic treatment.
IBS may present with a range of symptoms. However, abdominal pain and altered
bowel habits remain the primary features. Abdominal discomfort is often
described as crampy
86
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
in nature and located in the left lower quadrant, although the severity and
location can differ
greatly. Patients may report diarrhea, constipation, or alternating episodes
of diarrhea and
constipation. Diarrheal symptoms are typically described as small-volume,
loose stools, and
stool is sometimes accompanied by mucus discharge. Patients also may report
bloating, fecal
urgency, incomplete evacuation, and abdominal distention. Upper
gastrointestinal symptoms,
such as gastroesophageal reflux, dyspepsia, or nausea, may also be present
(Lynn and
Friedman, 1993).
Persistence of symptoms is not an indication for further testing; it is a
characteristic of
IBS and is itself an expected symptom of the syndrome. More extensive
diagnostic evaluation
is indicated in patients whose symptoms are worsening or changing. Indications
for further
testing also include presence of alarm symptoms, onset of symptoms after age
50, and a
family history of colon cancer. Tests may include colonoscopy, computed
tomography of the
abdomen and pelvis, and barium studies of the small or large intestine.
R. Sjiigren's Syndrome
The compounds and methods of this invention may be used for treating patients
with
SS. Primary Sjogren's syndrome (SS) is a chronic, slowly progressive, systemic
autoimmune
disease, which affects predominantly middle-aged women (female-to-male ratio
9:1),
although it can be seen in all ages including childhood (Jonsson et al.,
2002). It is
characterized by lymphocytic infiltration and destruction of the exocrine
glands, which are
infiltrated by mononuclear cells including CD4+, CD8+ lymphocytes and B-cells
(Jonsson et
al., 2002). In addition, extraglandular (systemic) manifestations are seen in
one-third of
patients (Jonsson et al., 2001).
The glandular lymphocytic infiltration is a progressive feature (Jonsson et
al., 1993),
which, when extensive, may replace large portions of the organs.
Interestingly, the glandular
infiltrates in some patients closely resemble ectopic lymphoid microstructures
in the salivary
glands (denoted as ectopic germinal centers) (Salomonsson et al., 2002;
Xanthou et al.,
2001). In SS, ectopic GCs are defined as T and B cell aggregates of
proliferating cells with a
network of follicular dendritic cells and activated endothelial cells. These
GC-like structures
formed within the target tissue also portray functional properties with
production of
autoantibodies (anti-Ro/SSA and anti-La/SSB) (Salomonsson and Jonsson, 2003).
In other systemic autoimmune diseases, such as RA, factors critical for
ectopic GCs
have been identified. Rheumatoid synovial tissues with GCs were shown to
produce
87
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
chemokines CXCL13, CCL21 and lymphotoxin (LT)- 13 (detected on follicular
center and
mantle zone B cells). Multivariate regression analysis of these analytes
identified CXCL13
and LT-I3 as the solitary cytokines predicting GCs in rheumatoid synovitis
(Weyand and
Goronzy, 2003). Recently CXCL13 and CXCR5 in salivary glands has been shown to
play
an essential role in the inflammatory process by recruiting B and T cells,
therefore
contributing to lymphoid neogenesis and ectopic GC formation in SS
(Salomonsson et al.,
2002).
S. Psoriasis
The compounds and methods of this invention may be used for treating patients
with
psoriasis. Psoriasis is a chronic skin disease of scaling and inflammation
that affects 2 to 2.6
percent of the United States population, or between 5.8 and 7.5 million
people. Although the
disease occurs in all age groups, it primarily affects adults. It appears
about equally in males
and females. Psoriasis occurs when skin cells quickly rise from their origin
below the surface
of the skin and pile up on the surface before they have a chance to mature.
Usually this
movement (also called turnover) takes about a month, but in psoriasis it may
occur in only a
few days. In its typical form, psoriasis results in patches of thick, red
(inflamed) skin covered
with silvery scales. These patches, which are sometimes referred to as
plaques, usually itch or
feel sore. They most often occur on the elbows, knees, other parts of the
legs, scalp, lower
back, face, palms, and soles of the feet, but they can occur on skin anywhere
on the body.
The disease may also affect the fingernails, the toenails, and the soft
tissues of the genitals
and inside the mouth. While it is not unusual for the skin around affected
joints to crack,
approximately 1 million people with psoriasis experience joint inflammation
that produces
symptoms of arthritis. This condition is called psoriatic arthritis.
Psoriasis is a skin disorder driven by the immune system, especially involving
a type
of white blood cell called a T cell. Normally, T cells help protect the body
against infection
and disease. In the case of psoriasis, T cells are put into action by mistake
and become so
active that they trigger other immune responses, which lead to inflammation
and to rapid
turnover of skin cells. In about one-third of the cases, there is a family
history of psoriasis.
Researchers have studied a large number of families affected by psoriasis and
identified genes
linked to the disease. People with psoriasis may notice that there are times
when their skin
worsens, then improves. Conditions that may cause flareups include infections,
stress, and
changes in climate that dry the skin. Also, certain medicines, including
lithium and beta
88
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
blockers, which are prescribed for high blood pressure, may trigger an
outbreak or worsen the
disease.
T. Infectious diseases
Compounds of the present disclosure may be useful in the treatment of
infectious
diseases, including viral and bacterial infections. As noted above, such
infections may be
associated with severe localized or systemic inflammatory responses. For
example, influenza
may cause severe inflammation of the lung and bacterial infection can cause
the systemic
hyperinflammatory response, including the excessive production of multiple
inflammatory
cytokines, that is the hallmark of sepsis. In addition, compounds of the
invention may be
useful in directly inhibiting the replication of viral pathogens. Previous
studies have
demonstrated that related compounds such as CDDO can inhibit the replication
of HIV in
macrophages (Vazquez et al., 2005). Other studies have indicated that
inhibition of NF-
kappa B signaling may inhibit influenza virus replication, and that
cyclopentenone
prostaglandins may inhibit viral replication (e.g., Mazur et al., 2007; Pica
et al., 2000).
VI. Pharmaceutical Formulations and Routes of Administration
The compounds of the present disclosure may be administered by a variety of
methods, e.g., orally or by injection (e.g. subcutaneous, intravenous,
intraperitoneal, etc.).
Depending on the route of administration, the active compounds may be coated
in a material
to protect the compound from the action of acids and other natural conditions
which may
inactivate the compound. They may also be administered by continuous
perfusion/infusion of
a disease or wound site.
To administer the therapeutic compound by other than parenteral
administration, it
may be necessary to coat the compound with, or co-administer the compound
with, a material
to prevent its inactivation. For example, the therapeutic compound may be
administered to a
patient in an appropriate carrier, for example, liposomes, or a diluent.
Pharmaceutically
acceptable diluents include saline and aqueous buffer solutions. Liposomes
include water-in-
oil-in-water CGF emulsions as well as conventional liposomes (Strejan et al.,
1984).
The therapeutic compound may also be administered parenterally,
intraperitoneally,
intraspinally, or intracerebrally. Dispersions can be prepared in glycerol,
liquid polyethylene
glycols, and mixtures thereof and in oils. Under ordinary conditions of
storage and use, these
preparations may contain a preservative to prevent the growth of
microorganisms.
89
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
Pharmaceutical compositions suitable for injectable use include sterile
aqueous
solutions (where water soluble) or dispersions and sterile powders for the
extemporaneous
preparation of sterile injectable solutions or dispersion. In all cases, the
composition must be
sterile and must be fluid to the extent that easy syringability exists. It
must be stable under the
conditions of manufacture and storage and must be preserved against the
contaminating
action of microorganisms such as bacteria and fungi. The carrier can be a
solvent or
dispersion medium containing, for example, water, ethanol, polyol (such as,
glycerol,
propylene glycol, and liquid polyethylene glycol, and the like), suitable
mixtures thereof, and
vegetable oils. The proper fluidity can be maintained, for example, by the use
of a coating
such as lecithin, by the maintenance of the required particle size in the case
of dispersion and
by the use of surfactants. Prevention of the action of microorganisms can be
achieved by
various antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be preferable
to include isotonic
agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol
and sorbitol,
in the composition. Prolonged absorption of the injectable compositions can be
brought
about by including in the composition an agent which delays absorption, for
example,
aluminum monostearate or gelatin.
Sterile injectable solutions can be prepared by incorporating the therapeutic
compound in the required amount in an appropriate solvent with one or a
combination of
ingredients enumerated above, as required, followed by filtered sterilization.
Generally,
dispersions are prepared by incorporating the therapeutic compound into a
sterile carrier
which contains a basic dispersion medium and the required other ingredients
from those
enumerated above. In the case of sterile powders for the preparation of
sterile injectable
solutions, the preferred methods of preparation are vacuum drying and freeze-
drying which
yields a powder of the active ingredient (i.e., the therapeutic compound) plus
any additional
desired ingredient from a previously sterile-filtered solution thereof.
The therapeutic compound can be orally administered, for example, with an
inert
diluent or an assimilable edible carrier. The therapeutic compound and other
ingredients may
also be enclosed in a hard or soft shell gelatin capsule, compressed into
tablets, or
incorporated directly into the subject's diet. For oral therapeutic
administration, the
therapeutic compound may be incorporated with excipients and used in the form
of ingestible
tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups,
wafers, and the like.
The percentage of the therapeutic compound in the compositions and
preparations may, of
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
course, be varied. The amount of the therapeutic compound in such
therapeutically useful
compositions is such that a suitable dosage will be obtained.
It is especially advantageous to formulate parenteral compositions in dosage
unit form
for ease of administration and uniformity of dosage. Dosage unit form as used
herein refers
to physically discrete units suited as unitary dosages for the subjects to be
treated; each unit
containing a predetermined quantity of therapeutic compound calculated to
produce the
desired therapeutic effect in association with the required pharmaceutical
carrier. The
specification for the dosage unit forms of the invention are dictated by and
directly dependent
on (a) the unique characteristics of the therapeutic compound and the
particular therapeutic
effect to be achieved, and (b) the limitations inherent in the art of
compounding such a
therapeutic compound for the treatment of a selected condition in a patient.
The therapeutic compound may also be administered topically to the skin, eye,
or
mucosa. Alternatively, if local delivery to the lungs is desired the
therapeutic compound may
be administered by inhalation in a dry-powder or aerosol formulation.
Active compounds are administered at a therapeutically effective dosage
sufficient to
treat a condition associated with a condition in a patient. A "therapeutically
effective
amount" preferably reduces the amount of symptoms of the condition in the
infected patient
by at least about 20%, more preferably by at least about 40%, even more
preferably by at
least about 60%, and still more preferably by at least about 80% relative to
untreated subjects.
For example, the efficacy of a compound can be evaluated in an animal model
system that
may be predictive of efficacy in treating the disease in humans, such as the
model systems
shown in the examples and drawings.
The actual dosage amount of a compound of the present disclosure or
composition
comprising a compound of the present disclosure administered to a subject may
be
determined by physical and physiological factors such as age, sex, body
weight, severity of
condition, the type of disease being treated, previous or concurrent
therapeutic interventions,
idiopathy of the subject and on the route of administration. These factors may
be determined
by a skilled artisan. The practitioner responsible for administration will
typically determine
the concentration of active ingredient(s) in a composition and appropriate
dose(s) for the
individual subject. The dosage may be adjusted by the individual physician in
the event of
any complication.
An effective amount typically will vary from about 0.001 mg/kg to about 1,000
mg/kg, from about 0.01 mg/kg to about 750 mg/kg, from about 100 mg/kg to about
500
91
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
mg/kg, from about 1.0 mg/kg to about 250 mg/kg, from about 10.0 mg/kg to about
150 mg/kg
in one or more dose administrations daily, for one or several days (depending,
of course, of
the mode of administration and the factors discussed above). Other suitable
dose ranges
include 1 mg to 10,000 mg per day, 100 mg to 10,000 mg per day, 500 mg to
10,000 mg per
day, and 500 mg to 1,000 mg per day. In some particular embodiments, the
amount is less
than 10,000 mg per day with a range, for example, of 750 mg to 9,000 mg per
day.
The effective amount may be less than 1 mg/kg/day, less than 500 mg/kg/day,
less
than 250 mg/kg/day, less than 100 mg/kg/day, less than 50 mg/kg/day, less than
25
mg/kg/day or less than 10 mg/kg/day. It may alternatively be in the range of 1
mg/kg/day to
200 mg/kg/day. For example, regarding treatment of diabetic patients, the unit
dosage may
be an amount that reduces blood glucose by at least 40% as compared to an
untreated subject.
In another embodiment, the unit dosage is an amount that reduces blood glucose
to a level
that is 10% of the blood glucose level of a non-diabetic subject.
In other non-limiting examples, a dose may also comprise from about 1
microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 micro-
gram/kg/body weight, about 50 microgram/kg/body weight, about 100
microgram/kg/body
weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body
weight, about
500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5
milligram/kg/body weight, about 10 milligram/kg/body weight, about 50
milligram/kg/body
weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body
weight, about 350
milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1,000
mg/kg/body
weight or more per administration, and any range derivable therein. In non-
limiting examples
of a derivable range from the numbers listed herein, a range of about 5
mg/kg/body weight to
about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500
milligram/kg/body weight, etc., can be administered, based on the numbers
described above.
In certain embodiments, a pharmaceutical composition of the present disclosure
may
comprise, for example, at least about 0.1% of a compound of the present
disclosure. In other
embodiments, the compound of the present disclosure may comprise between about
2% to
about 75% of the weight of the unit, or between about 25% to about 60%, for
example, and
any range derivable therein.
Single or multiple doses of the agents are contemplated. Desired time
intervals for
delivery of multiple doses can be determined by one of ordinary skill in the
art employing no
more than routine experimentation. As an example, subjects may be administered
two doses
92
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
daily at approximately 12 hour intervals. In some embodiments, the agent is
administered
once a day.
The agent(s) may be administered on a routine schedule. As used herein a
routine
schedule refers to a predetermined designated period of time. The routine
schedule may
encompass periods of time which are identical or which differ in length, as
long as the
schedule is predetermined. For instance, the routine schedule may involve
administration
twice a day, every day, every two days, every three days, every four days,
every five days,
every six days, a weekly basis, a monthly basis or any set number of days or
weeks there-
between. Alternatively, the predetermined routine schedule may involve
administration on a
twice daily basis for the first week, followed by a daily basis for several
months, etc. In other
embodiments, the invention provides that the agent(s) may taken orally and
that the timing of
which is or is not dependent upon food intake. Thus, for example, the agent
can be taken
every morning and/or every evening, regardless of when the subject has eaten
or will eat.
VII. Combination Therapy
In addition to being used as a monotherapy, the compounds of the present
disclosure
may also find use in combination therapies. Effective combination therapy may
be achieved
with a single composition or pharmacological formulation that includes both
agents, or with
two distinct compositions or formulations, at the same time, wherein one
composition
includes a compound of this invention, and the other includes the second
agent(s).
Alternatively, the therapy may precede or follow the other agent treatment by
intervals
ranging from minutes to months.
Various combinations may be employed, such as when a compound of the present
disclosure is "A" and "B" represents a secondary agent, non-limiting examples
of which are
described below:
A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B
B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A
B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A
Administration of the compounds of the present disclosure to a patient will
follow
general protocols for the administration of pharmaceuticals, taking into
account the toxicity,
if any, of the drug. It is expected that the treatment cycles would be
repeated as necessary.
93
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
Beta interferons may be suitable secondary agents. These are medications
derived
from human cytokines which help regulate the immune system. They include
interferon 13-lb
and interferon 13-1a. Betaseron has been approved by the FDA for relapsing
forms of
secondary progressive MS. Furthermore, the FDA has approved the use of several
13-interferons as treatments for people who have experienced a single attack
that suggests
multiple sclerosis, and who may be at risk of future attacks and developing
definite MS. For
example, risk of MS may be suggested when an MRI scan of the brain shows
lesions that
predict a high risk of conversion to definite MS.
Glatiramer acetate is a further example of a secondary agent that may be used
in a
combination treatment. Glatiramer is presently used to treat relapsing
remitting MS. It is
made of four amino acids that are found in myelin. This drug is reported to
stimulate T cells
in the body's immune system to change from harmful, pro-inflammatory agents to
beneficial,
anti-inflammatory agents that work to reduce inflammation at lesion sites.
Another potential secondary agent is mitoxantrone, a chemotherapy drug used
for
many cancers. This drug is also FDA-approved for treatment of aggressive forms
of relapsing
remitting MS, as well as certain forms of progressive MS. It is given
intravenously, typically
every three months. This medication is effective, but is limited by cardiac
toxicity.
Novantrone has been approved by the FDA for secondary progressive, progressive-
relapsing,
and worsening relapsing-remitting MS.
Another potential secondary agent is natalizumab. In general, natalizumab
works by
blocking the attachment of immune cells to brain blood vessels, which is a
necessary step for
immune cells to cross into the brain, thus reducing the immune cells'
inflammatory action on
brain neurons. Natalizumab has been shown to significantly reduce the
frequency of attacks
in people with relapsing MS.
In the case of relapsing remitting MS, patients may be given intravenous
corticosteroids, such as methylprednisolone, as a secondary agent, to end the
attack sooner
and leave fewer lasting deficits.
Other common drugs for MS that may be used in combination with the present
oleanolic acid derivatives include immunosuppressive drugs such as
azathioprine, cladribine
and cyclophosphamide.
It is contemplated that other anti-inflammatory agents may be used in
conjunction
with the treatments of the current invention. Other COX inhibitors may be
used, including
arylcarboxylic acids (salicylic acid, acetylsalicylic acid, diflunisal,
choline magnesium
94
CA 02721837 2015-07-30
trisalicylate, salicylate, benorylate, flufenamic acid, mefenamic acid,
meclofenamic acid and
triflumic acid), arylalkanoic acids (diclofenac, fenclofenac, alclofenac,
fentiazac, ibuprofen,
flurbiprofen, ketoprofen, naproxen, fenoprofen, fenbufen, suprofen,
indoprofen, tiaprofenic
acid, benoxaprofen, pirprofen, tolmetin, zomepirac, clopinac, indomethacin and
sulindac) and
enolie acids (phenylbutazone, oxyphenbutazone, azapropazone, feprazone,
piroxicam, and
isoxicam. See also U.S. Pat. No. 6,025,395.
Histamine H2 receptor blocking agents may also be used in conjunction with the
compounds of the current invention, including cimetidine, ranitidine,
famotidine and
nizatidine.
Treatment with acetylcholinesterase inhibitors such as tacrine, donepizil,
metrifonate
and rivastigmine for the treatment of Alzheimer's and other disease in
conjunction with the
compounds of the present disclosure is contemplated. Other
acetylcholinesterase inhibitors
may be developed which may be used once approved include rivastigminc and
metrifonatc.
Acetylcholinesterase inhibitors increase the amount of neurotransmitter
acetylcholine at the
nerve terminal by decreasing its breakdown by the enzyme cholinesterase.
MAO-B inhibitors such as selegilene may be used in conjunction with the
compounds
of the current invention. Selegilene is used for Parkinson's disease and
irreversibly inhibits
monoamine oxidase type B (MAO-B). Monoamine oxidase is an enzyme that
inactivates the
monoamine neurotransmitters norepinephrine, serotonin and dopamine.
Dietary and nutritional supplements with reported benefits for treatment or
prevention
of Parkinson's, Alzheimer's, multiple sclerosis, amyotrophic lateral
sclerosis, rheumatoid
arthritis, inflammatory bowel disease, and all other diseases whose
pathogenesis is believed
to involve excessive production of either nitric oxide (NO) or prostaglandins,
such as acetyl-
L-carnitine, octacosanol, evening primrose oil, vitamin B6, tyrosine,
phenylalanine, vitamin
C, L-dopa, or a combination of several antioxidants may be used in conjunction
with the
compounds of the current invention.
For the treatment or prevention of cancer, compounds of the invention may be
combined with one or more of the following: radiation, chemotherapy agents
(e.g., cytotoxic
agents such as anthracyclines, vincristine, vinblastin, microtubule-targeting
agents such as
paclitaxel and docetaxel, 5-FU and related agents, cisplatin and other
platinum-containing
compounds, irinotecan and topotecan, gemcitabine, temozolomide, etc.),
targeted therapies
(e.g., imatinib, bortezomib, bevacizumab, rituximab), or vaccine therapies
designed to
promote an enhanced immune response targeting cancer cells.
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
For the treatment or prevention of autoimmune disease, compounds of the
invention
may be combined with one or more of the following: corticosteroids,
methotrexate, anti-TNF
antibodies, other TNF-targeting protein therapies, and NSAIDs. For the
treatment of
prevention of cardiovascular diseases, compounds of the invention may be
combined with
antithrombotic therapies, anticholesterol therapies such as statins (e.g.,
atorvastatin), and
surgical interventions such as stenting or coronary artery bypass grafting.
For the treatment
of osteoporosis, compounds of the invention may be combined with
antiresorptive agents
such as bisphosphonates or anabolic therapies such as teriparatide or
parathyroid hormone.
For the treatment of neuropsychiatric conditions, compounds of the invention
may be
combined with antidepressants (e.g., imipramine or SSRIs such as fluoxetine),
antipsychotic
agents (e.g., olanzapine, sertindole, risperidone), mood stabilizers (e.g.,
lithium, valproate
semisodium), or other standard agents such as anxiolytic agents. For the
treatment of
neurological disorders, compounds of the invention may be combined with
anticonvulsant
agents (e.g., valproate semisodium, gabapentin, phenytoin, carbamazepine, and
topiramate),
antithrombotic agents (e.g., tissue plasminogen activator), or analgesics
(e.g., opioids, sodium
channel blockers, and other antinociceptive agents).
For the treatment of disorders involving oxidative stress, compounds of the
present
disclosure may be combined with tetrahydrobiopterin (BH4) or related
compounds. BH4 is a
cofactor for constitutive forms of nitric oxide synthase, and may be depleted
by reactions with
peroxynitrite. Peroxynitrite is formed by the reaction of nitric oxide and
superoxide. Thus,
under conditions of oxidative stress excessive levels of superoxide can
deplete normal,
beneficial levels of nitric oxide by converting NO to peroxynitrite. The
resulting depletion of
BH4 by reaction with peroxynitrite results in the "uncoupling" of nitric oxide
synthases so
that they form superoxide rather than NO. This adds to the oversupply of
superoxide and
prolongs the depletion of NO. Addition of exogenous BH4 can reverse this
uncoupling
phenomenon, restoring the production of NO and reducing the level of oxidative
stress in
tissues. This mechanism is expected to complement the actions of compounds of
the
invention, which reduce oxidative stress by other means, as discussed above
and throughout
this invention.
VIII. Examples
The following examples are included to demonstrate preferred embodiments of
the
invention. It should be appreciated by those of skill in the art that the
techniques disclosed in
96
CA 02721837 2015-07-30
the examples which follow represent techniques discovered by the inventor to
function well
in the practice of the invention, and thus can be considered to constitute
preferred modes for
its practice.
Example 1 ¨ Methods and Materials
Nitric Oxide production and cell viability. RAW264.7 macrophages were pre-
treated with DMSO or drugs for 2 hours, then treated with recombinant mouse
IF.Ny (Sigma)
for 24 hours. NO concentration in media was determined using the Griess
reagent system
(Promega). Cell viability was determined using WST-1 reagent (Roche).
STAT3 phosphorylation. HeLa cells were treated with the indicated compounds
and
concentrations for 6 hours and subsequently stimulated with 20 ng/ml
recombinant human IL-
6 (R&D Systems) for 15 minutes. Lysates were immunoblotted with antibodies
against
phosphorylated or total STAT3 (Cell Signaling).
NF-KB activation. HeLa cells were transfected with pNF-KB-Luc (inducible,
Stratagene) and pRL-TK (constitutive, Promega) reporter plasmids. Twenty-four
hours later
cells were pre-treated with the indicated compounds for 2 hours. DMSO served
as a vehicle
control. Following pre-treatment, cells were stimulated with 20 ng/ml
recombinant human
TNFa (BD Biosciences) for 3 hours. Reporter activity was measured using
DualGlo
lueiferase reporter system (Promega) and pNF-KB luciferase activity was
normalized against
pRL-TK luciferase activity.
Fold-induction of mean tuciferase activity relative to
unstimulated (-TNFa) samples is shown. Error bars represent the SD of the mean
of 6
samples.
lxBa degradation. HeLa cells were treated with indicated compounds and
concentrations for 6 hours and subsequently stimulated with 20 ng/ml TNFa for
15 minutes.
Lysates were blotted with antibodies against IKBa (Santa Cruz) and actin
(Chemicon).
COX-2 induction Western blot. RAW264.7 cells were pre-treated for 2 hours with
indicated compounds and subsequently stimulated with 10 ng/ml IFNy for an
additional 24
hours. COX-2 protein levels were assayed by immunoblotting using an antibody
from Santa
Cruz. Actin was used as a loading control.
97
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
Nrf2 target gene induction. MDA-MB-435 human melanoma cells were treated
with vehicle (DMSO) or the indicated compounds and concentrations for 16
hours. HO-1,
thioredoxin reductase-1 (TrxR1), y-glutamylcysteine synthetase (y-GCS), and
ferritin heavy
chain mRNA levels were quantified using qPCR and were normalized relative to a
DMS0-
treated sample run in parallel. Values are averages of duplicate wells. Primer
sequences are
as follows.
HO-1 FW: TCCGATGGGTCCTTACACTC (SEQ ID NO:1),
HO-1 REV: TAGGCTCCTTCCTCCTTTCC (SEQ ID NO:2),
TrxR1 FW: GCAGCACTGAGTGGTCAAAA (SEQ ID NO:3),
TrxR1 REV: GGTCAACTGCCTCAATTGCT (SEQ ID NO:4),
y-GCS FW: GCTGTGGCTACTGCGGTATT (SEQ ID NO:5),
y-GCS REV ATCTGCCTCAATGACACCAT (SEQ ID NO:6),
Ferritin HC FW: ATGAGCAGGTGAAAGCCATC (SEQ ID NO:7),
Ferritin HC REV: TAAAGGAAACCCCAACATGC (SEQ ID NO:8),
S9 FW: GATTACATCCTGGGCCTGAA (SEQ ID NO:9),
S9 REV: GAGCGCAGAGAGAAGTCGAT (SEQ ID NO:10).
Comparison Compounds. Some of the experimental results presented below and
throughout this application present data for not only the compounds discussed
above, but also
for one or more of the triterpenoid derivatives shown in the table below.
0
gidr 0
NC ar,W 0 NC
OH
0,CH3
0
0 7F-iw 0 7,,
401 (RTA 401) 402 (RTA 402)
0
0
0
NC
Fl--- NC CH3 00 NH2
gob
0 7F-iw 0 rr
402-19 402-52
98
CA 02721837 2015-07-30
.,:' :
.-
0 O 0 0
o 0
NCN-S-:CH3
a= NC it&PIIIII
WV OH
402-53 402-54
.=-'' .,,s'
0 0 r o 110
00
NC so i NC .7AmWple CN
0
0 - ,--,-
402-55 402-56
0 1: 1V1P0 4
110 HN
eO.ri
0
4
NC70L2 NC540
-- --,
404 (RTA 404) 63112
=,,,,,
= illp
410
OH3
H
OS N. OS N.0,CH3
CH3
NC igkiih NC gaigh=
.:
0 0
0 P..-1. 0 qUPP.--711P
63324 63166
Several of the above compounds, including 401, 402, 402-56 and 404 can be
prepared according to the methods taught by Honda et al. (1998), Honda et al.
(2000b),
Honda et al. (2002), Yates et al. (2007), U.S. Patent 6,974,801, and U.S.
Applications
Publications 2010-0048892, 2010-0048911 and 2010-0041904. The synthesis of the
other
compounds may be prepared according to the methods disclosed in one or more of
the
following applications filed concurrently herewith: U.S. Patent Application
99
CA 02721837 2015-07-30
Publication No. 2010-0048892 by Eric Anderson, Xin Jiang and Melean Visnick,
entitled
**Antioxidant Inflammation Modulators: Oleanolic Acid Derivatives with Amino
and Other
Modifications At C-17," filed April 20, 2009; U.S. Patent Application
Publication No. 2010-
0048911 by Xin Jiang, Jack Greiner, Lester Maravetz, Stephen S. Szucs, Mclean
Visnick, entitled
"Antioxidant Inflammation Modulators: Novel Derivatives of Oleanolic Acid,"
filed April 20,
2009; U.S. Patent Application Publication No. 2010-0041904 by Xin Jiang,
Xiaofeng Liu, Jack
Greiner, Stephen S. Szucs, Melean Visnick entitled, "Antioxidant Inflammation
Modulators: C-
17 1-lomologated Oleanolic Acid Derivatives," filed April 20, 2009.
Aqueous Solubility, Determination. The following procedure was used to obtain
the
aqueous solubility results summarized in Example 8. Step 1. Determination of
optimal
UV/vis wavelengths and generation of standard curves for a compound of
interest:
(1) For eight standard calibration curves (one plate), prepare 34 mL of
50:50 (v:v)
universal bufferacctonitrile in a 50 nth tube.
(2) Using a multichannel pipet, dispense (in L) the buffer:acetonitrile in
a deep
well plate as follows:
1 2 3 4 5 6 7 8 9 10 11 12
A
285 285 380 380 285 285 285 285 285 285 285 285
(3) Using a multichannel pipet, dispense DMSO into the same plate as
follows:
1 2 3 4 5 6 7 8 9 10 11 12
A
12 12 15 15 15 15 15 15 15 15
tiL L uL tL L ,uL L ,uL )at
100
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
(4) Add 10 mM compound in DMSO into the plates as follows:
1 2 3 4 5 6 7 8 9 10 11 12
A 15 L 15 L 8 L 8 L
cmpd1 cmpd1 cmpd1 cmpd1
B 15 L 15 L 8 L 8 L
cmpd2 cmpd2 cmpd2 cmpd2
C 15 L 15 L 8 L 8 L
cmpd3 cmpd3 cmpd3 cmpd3
D 15 L 15 L 8 L 8 L
cmpd4 cmpd4 cmpd4 cmpd4
15 L 15 L 8 L 8 L
E
cmpd5 cmpd5 cmpd5 cmpd5
F 15 L 15 L 8 L 8 L
cmpd6 cmpd6 cmpd6 cmpd6
G 15 L 15 L 8 L 8 L
cmpd7 cmpd7 cmpd7 cmpd7
H 15 L 15 L 8 L 8 L
cmpd8 cmpd8 cmpd8 cmpd8
(5) Mix columns 1 and 2 by pipetting each up and down 10 times.
Mix columns 3
and 4 by pipetting up and down 10 times. Serially dilute as follows (pipet up
and down 10 times after each transfer):
100 u 100 !I 100 u
1 2 3 4 5 6 7 8 9 10 11 12
A
B
C
D
E
F
G
H
Note columns 11 and 12 contain DMSO only and so compound should not be
transferred to these wells.
(6) Cover plate with lid and shake (200-300 rpm) at room temperature for 20
minutes.
(7) Mix all wells by pipetting up and down 10 times.
101
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
(8) Transfer 120 uL from each well to a UV transparent plate. Cover and
shake for
3-5 minutes. Remove any bubbles in the wells using a pipet.
(9) Read from 220 nm to 500 nm at 10 nm increments on a spectrophotometer
(e.g., SpectraMax0).
Step 2. Compound Solubility Testing Procedures using the MilliporeTM
Multiscreen0
Solubility Filter Plate.
Consumables: MilliporeTM Multiscreen0 Solubility Filter Plate #MSSLBPC10
Greiner 96 well disposable UV-Star analysis plate, VWR#655801
Greiner 96 well polypropylene V-bottom collection plate, VWR#651201
Universal Aqueous Buffer:
(a) To prepare 500 mL of universal buffer, add the
following: 250 mL
Nanopure water; 1.36 mL (45 mM) ethanolamine; 3.08 g (45 mM)
potassium dihydrogen phosphate; 2.21 g (45 mM) potassium acetate;
thoroughly mix.
(b) Adjust pH to 7.4 with HC1 and q.s. to 500 mL with 0.15 M KC1.
(c) Filter to remove particulates and reduce bacterial growth.
(d) Store at 4 C in the dark.
Solubility Protocol:
(a) Add 285 uL of Universal Aqueous Buffer to desired wells of the
MilliporeTM Multiscreen0 Solubility filter plate.
(b) Add 15 uL of 10 mM compound in DMSO to the appropriate wells.
Add 15 uL of 100% DMSO only to 6 wells of the filter plate for
blanks.
(c) Using a multichannel pipet, mix wells by pipetting up and down 10
times. Be careful not to touch the filters in the plate with the tips.
(d) Cover and gently shake (200-300 rpm) filter plate for 90 minutes at
room temperature.
(e) Vacuum filter the aqueous solution from the Multiscreen0 solubility
filter plate into a polypropylene V-bottom plate.
(f) Transfer 60 uL of filtrate to a UV transparent plate (Greiner UV-Star
Analysis Plate).
102
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
(g) Add 60 L of acetonitrile to each well and mix by pipetting up and
down 10 times.
(h) Cover and gently shake for 3-5 minutes. Remove any bubbles with a
pipet.
(0 Measure the absorbance of each well in the plate on the
spectrophotometer (UV/vis) at the desired wavelength. For compounds
in a plate with different absorbance peaks, set the spectrophotometer to
read a spectrum (e.g., from 220 nm to 460 nm).
(i) Identify concentration using measured absorbance for each compound
and the predetermined standard curve (see Step 1).
Example 2 ¨ Synthesis of Oleanolic Acid Derivatives
The synthesis of compounds 402-02 and 402-51 started from compound 1 (Scheme
1).
Compound 1 was oxidized with bleach to give ketone 2 in 80% yield. Formylation
of 2 with
ethyl formate using sodium methoxide as the base afforded compound 402-48 (70%
yield),
which was then treated with hydroxylamine hydrochloride in aqueous Et0H at 55
C to give
isoxazole 402-49 in 93% yield. Cleavage of the isoxazole under basic
conditions gave a-
cyanoketone 402-46 in quantitative yield as a mixture of ketone and enol
forms. Compound
402-46 was treated with 1,3-dibromo-5,5-dimethylhydantoin, followed by
elimination of HBr
using pyridine as the base, to give compound 402-02 in 81% yield (from 402-
49), which was
demethylated with LiI in refluxing DMF to give acid 402-51 in 95% yield.
103
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
Q)
2 =
11 II
rI ce ce
0
C / IN r 9
.
0 9 1
a) .,,,
2 0 0
CT
I
0
/ õ * o = . li I
co
0 Tr
A
0
I
0 am
...., =4 0 0 II
0 4-
..,,
W
.,I
W // 0
z
I/
=0
al
14 a)
Q) 0
2 ci
CT ,,,, *
. CO
0
I 0 Tr
cDA
/,. .
. 1. . 1 .4.
= -C
1 1 CV
ill..
0
0
WO 1
0"1
*
..ii
// 2
z
0
(f
-ci
Q) a)
2 2
O' O'
0 0
õ,,.. õ,,..
I
IN
0
i ...11 r
I ...11 "4
0 am
1117 0 am
=11 1117
.1=
W
0
I N ,0
z
,,
cu
E
a.)
,4
cJ
cf)
104
CA 02721837 2010-10-15
WO 2009/129545 PCT/US2009/041170
Reagents and conditions applicable to Scheme 1 are: (a) AcOH, bleach, room
temperature, 1 h, 80%; (b) HCO2Et, Na0Me, 55 C, 24 h, 70%; (c) NH2OH=FIC1, 55
C, 16 h, 93%; (d) Na0Me, 55 C, 2 h, 100%; (e) (i) 1,3-dibromo-5,5-
dimethylhydantoin, room temperature, 2 h; (ii) pyridine, 55 C, 15 h, 81%; (f)
LiI,
160 C, 8 h, 95%.
Compound 402-63 was oxidized with Dess-Martin periodinane to give
aldehyde 402-64 in 47% yield (Scheme 2).
Scheme 2:
i
. H O H O
00 _
N a 0:0 0
OW OH IN OS
0 0
H 402-63
I-I- 402-64
Reagents and conditions applicable to Scheme 2 are: (a) Dess-Martin
periodinane, NaHCO3, room temperature, 1 h, 47%.
The synthesis of compounds 402-59 and 402-57 began from 402-51.
Compound 402-51 was treated with oxalyl chloride and catalytic DMF to give
acid
chloride 3. Compound 3 was treated with ammonia in methanol to give 402-59
(99%
from 402-51). Dehydration of 402-59 utilizing TFAA and Et3N gave dicyano
compound 402-57 (45% yield).
105
80535090.1
CA 02721837 2010-10-15
WO 2009/129545 PCT/US2009/041170
Scheme 3:
0 O 0 O
OH aCI b
_,..
NC .041)"
0 NC .041),
0
O I:1 0 I:1
402-51 3
0 O 0 0
N H 2 c
_,..
NC *se,
0 NC0 se" CN
O I:1 0 I:1
402-59 402-57
Reagents and conditions applicable to Scheme 3 are: (a) (C0C1)2, DMF (cat.),
0 C to room temperature, 3 h; (b) NH3 (Me0H), 0 C to room temperature, 5 h,
99%;
(c) TFAA, Et3N, 0 C, 3 h, 45%.
404-02 was synthesized from compound 3 as summarized in Scheme 4.
Compound 3 was reacted with 2,2,2-trifluoroethylamine-HC1 in toluene and water
at
70 C with NaHCO3 as the base, giving 404-02 in 69% yield.
Scheme 4:
0 O 000
H CI a 00O
N C F3
NC es i NC so=i
0 0
O I:I 0 I:1
3 404-02
Reagents and conditions applicable to Scheme 4 are: (a) 2,2,2-
trifluoroethylamine-HC1, NaHCO3, toluene, H20, 70 C, 69%.
106
80535090.1
CA 02721837 2010-10-15
WO 2009/129545 PCT/US2009/041170
a) a) a)
2
-.] 2
61 0 Cc)1
CD CD
/õ,.. o .õ,, .õ,.
.11 5
ii ...., . ...., ,.... o
ii ...., (...
0,..04w
W 0,.. CAFA 0" Gm
W =
C 1
IOW II
W
Nz-0 i/ 2 /, 0
z z
a) a) a)
2 2 2
61 61 61
CD CD
o/õ,.
.0 . // ' ' ' .0 //, , . *
(9
(9
-Jo- 0 N
0
_ el 11 = -9.- _(:) ii 0
. . 111 (9 (i ..111
i_T 71-
6 am
0
.oli 0
0
oil W
1 1 i
W W W
,0 // 2 /, 0
z z z
col
.
2
1
o
o/,..
õ
11 0)
711 -
C\I
0
1 ..111 71-
0
0
.'I
\z,C)
iii
a.)
E
cl.)
,4
CO
cf)
107
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
Reagents and conditions applicable to Scheme 5 are: (a) LiA1H4, THF, 0 C, 1.5
h,
38% for 4; 34% for 5; (b) Na0Me, 55 C, 7 h, 94% for 6; 89% for 7; (c) (i) 1,3-
dibromo-5,5-
dimethylhydantoin, r.t., 40 min; (ii) pyridine, 55 C, 25 h, 35% for 402-66;
32% for 63219.
The synthesis of 402-66 began from isoxazole compound 402-49. Reduction of the
ketone in 402-49 was achieved by treatment with LiA1H4 in THF at 0 C, giving
compounds
4 and 5 (as a 1:1 mixture of diastereotopic alcohols). Compound 4 was treated
with Na0Me
in Me0H at 55 C to give 6, which exists as a 3:2 mixture of keto and enol
tautomeric forms.
Bromination and subsequent dehydrobromination of 6 by treatment with 1,3-
dibromo-5,5-
dimethylhydantoin and then pyridine gave 402-66 in 33% yield (from 4). Using
the same
synthetic sequence, compound 5 was converted into 63219 in 28% overall yield.
80535090.1 108
CA 02721837 2010-10-15
WO 2009/129545 PCT/US2009/041170
C:)
,z4
iz 0
,,,,.. 0
0 cr)
i ....,
0 CALft
W i
.I
Z
i/ 0
\
z 0
õ,,, 1====
CO
0 t WO cC4
CO
C\I i =Iiil
i
Z 0
8
ZZ
11 CO
...II 0
= 00 z
= 10
// ,z4
iz 0
z
(NI
CO NA [10)
0
.%-
0 it, 0
/õ,.. t.õ4
Ipli
1 0
..111 CO
Cr
0 aft
117 i Z
111 i
-0 1
Z
'..C.)
cl.)
E
cl.)
=
CO
Cf)
109
CA 02721837 2010-10-15
WO 2009/129545 PCT/US2009/041170
Reagent and conditions applicable to Scheme 6 are: (a) oxalyl chloride, r.t.,
2 h; (b)
NH2NH2-H20, 0 C, 30 min, 97%; (c) AcC1, Et3N, r.t., 1 h, 77%; (d) Na0Me,
r.t., 10 min,
72%; (e) Ts0H, 110 C, 1 h, 33%.
110
CA 02721837 2010-10-15
WO 2009/129545 PCT/US2009/041170
i i i
z, z. z,
Z' , e Z Z Z Z Z
\ II \ Ii \ il
' Z 'Z ' Z
/,µ,.. /,µ,.
S õõ.
*0 cr)
0
1 ..oil eNi
,
1 ....1 el
i
,
....1 eNi
eNi
el
o
= co -). 0 OA FA _ 0 IC
0
o
W ai
=
.il = .11
*
0 N0, // 0
z z
_al
/
2
C.)
i
z z
// z' 'z
\ ii
/õ,..z
.0
1 IS ,
, ,,, ,
0 , Tr
..111
CO 1 ..111
0 001-
..1 i
*
*
.11I
0
N ,0
z
4¨
col i
2
2 0
I
ZZ.
0 Z, Z
\ ii
,,,
,, . ' Z
0 o
, .
o
el
i ...li , =3 c4
I
el
0 OAK 0= ....1
00 u)
li
=.. 1 =
0
// 0
z
r:=:.
a.)
E
a.)
=
cJ
C/
111
CA 02721837 2010-10-15
WO 2009/129545 PCT/US2009/041170
Reagent and conditions applicable to Scheme 7 are: (a) Et3N, TFAA, r.t., 1.5
h, 85%; (b) Bu3SnN3, 150 C, 67%; (c) (i) HCO2Et, Na0Me, 0 C to room
temperature, 1 h; (ii) NH2OH=HC1, 60 C, 3 h, 54%; (d) Na0Me, 55 C, 2 h; (e)
1,3-
dibromo-5,5-dimethylhydantoin, r.t., 2 h; (f) pyridine, 55 C, 16 h, 60% for
63229;
69% for 63230; (g) TMSCHN2, 0 C, 10 min, 77%.
112
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
_at
u_ u_
/,õ, ei
2Z r=-=
u_ el
0 c.1... 1 1
,,,,, . 0 el
el u)
0 eNi
eN
el
u) LZ 0
00
i ...II ..ii
0 OALEA
*
..W*
1I
//
Z 0
i
N _0
z
o 1
co 1
u_ u_
u_ u_
iz
0
/õ...iz
0
0 r=-=
eN
eN
/õ, el
= a
In
,- 0 aft I ...11
(1)
13
W
0 OAK =iii
*
W
..1=
* //
Z 0
I
0
cieS
a.)
E
a.)
,4
cJ
C/
113
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
Reagents and conditions applicable to Scheme 8 are: (a) i) Na0Me, HCO2Et, 0 C
to
room temperature; concentrated HC1 ii) NH2OH-HC1, Et0H, H20, 60 C, 14 h, 47%;
(b)
Na0Me, 55 C, 16 h, quantitative; (c) (i) 1,3-dibromo-5,5-dimethylhydantoin,
room
temperature, 3 h; (ii) pyridine, 55 C, 16 h, 12%.
Scheme 9:
i ..
= H O (CF3 OHH O (CF3
NH NH
a b
AlOOD SO
N 1 IO le 1\1
0 ,1 100 - 0
0 0 _
I:1 63223 H 16
OHH O (CF3 OHH O (CF3
SID
N NH SO NH
c N
Ie. 0 OS 0
HO
:17 0 -
H 63268
Reagents and conditions applicable to Scheme 9 are: (a) LiA1H4, THF, 0 C, 2
h, 19%;
(b) Na0Me, 55 C, 7 h, 92%; (c) (i 1,3-dibromo-5,5-dimethylhydantoin, r.t., 50
min; (ii)
pyridine, 55 C, 7 h, 56%.
114
CA 02721837 2010-10-15
WO 2009/129545 PCT/US2009/041170
c.)
<
. i
z¨z
I
.. 0
= o
....1 cv
= aft
W
..-,-
..i_,_ _.õ.
z' y
zõõ.
, \
z' y ci)
\ ,õ,..
_ , o
õ,.. r-I
,0
0 cv
el
Z
0 cv
co 0 aWft
...II co
1DI0
IN .11=
0
0 ..00
/õ,..
0a) , C.) 0
Z
, 0
...11
0 Gm
W +Z (DI
Z
Z' /-----. Z' y
c
.z,o \ cn \c/)/õ.. = /õ...
coI = Ú¨ 0
el
...II cv ...II cv
-3.-
IN 0 Irro -o 0 Co
0
c.)
= co * . s % %% * , 0%
..111 ,-
0 N ,0 C.) 0
W z z
IDALEA i
..ii
0
O
,-1
a.)
E
a.)
=
c.)
cf)
115
CA 02721837 2015-07-30
Reagents and conditions pertaining to Scheme 10: (a) (i) HCO2Et, Na0Me, 0 C,
1.5
h; (ii) NH2OH-HC1, 65 C, 3.5 h, 78%; (b) (i) oxalyl chloride, 0 C to rt, 2
h; (ii) AcNHNI-I2,
Et3N, 0 C to rt, 30 min, 99%; (c) Lawesson's reagent, 110 C, 30 min, 10%
(for compound
21) and 29% (for compound 22); (d) Na0Me, 55 C, 2 h, 73%; (e) (i) DBDMH, 0
C, 1 h; (ii)
pyridine, 55 C, 3 h, 79%. Compound 18 was reported by Honda et al. (2000a).
116
Scheme 11:
0
OH OH
w H O Hap Hap g
O *0 0 C 02M e a H 0
OH b HO c 01)
- CHO _,-- r..) PAP PAP un
.i
un RIP
OREP
F Fl
HO i
Fi
1 24
25
0 H O OH
= n
H H O
H O
O. 00 / H 0 e
gi d
f o
n)
-=.-
_,.. .,1
C I
UJ
.,1
HO OAP
0 OgiP
H
1
27
28 0H
IL
(continued on next page)
.d
n
,-i
cpw
=
=
-a--,
.i
--.1
=
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
(continued from previous page):
0
H O 0
H O
OH g0-0 h
-----. -A-
40". -........ -A.
NI I O. -
0
0 -
IR H
29
H O H O
e.
NC 00 NC athei.
H H
31 63303
Reagents and conditions applicable to Scheme 11: (a) LAH, rt to 65 C, 1.5 h,
27%;
(b) TEMPO, IPh(OAc)2, rt, 72 h, 77%; (c) (Ph3PCH2C1)C1 / n-BuLi, THF, HMPA, 0
C to rt,
5 87%; (d) MeLi, THF, 0 C to rt, 91%; (e) PCC, Na0Ac, CH2C12, rt, 78%; (f)
HCO2Et,
Na0Me, 0 C to rt; (g) NH2ORHC1, Et0H-H20, 60 C, 89% from 28; (h) Na0Me, 55
C,
3 h; (i) DDQ, Benzene, 85 C, 39% from 30.
Scheme 12:
H O H O
H O
NC ra
=
a1" b
_,..
NC
HO 1-411. HO -
H A H
31 32
63308
10 Reagents and conditions applicable to Scheme 12 are: (a) HgSO4, H2SO4,
acetone/H20, 55 C, 20 h, 91%; (b) (i) 1,3-dibromo-5,5-dimethylhydantoin, 0
C, 3 h; (ii)
pyridine, 55 C, 19 h, 73%.
118
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
Scheme 13:
0 O
0 0 =
Adr 0 0
00 CO2H
NC digh . - NC der:W
).. 0
a
H
--- --- I:1
, -
402-51 63323
Reagents and conditions pertaining to Scheme 13: (a) benzyl bromide, DBU, 100
C,
6 h, 65%.
Scheme 14:
., .,
0 O 0 0
H
CI
NC 00 0 N,e
NC aire a iiii
0 -,..
0 -411F 0 -411F
----I:I ---,I-I
3 63325
Reagents and conditions pertaining to Scheme 14: (a) MeONH2-HC1, Et3N, 40 C,
4 h,
37%.
Scheme 15:
..,:
oó 0 0
I
SC CI SC I\1
NC . a NC alib i
0 ¨v.- 0
O,, 0 -41
"-- H
-
3 63326
Reagents and conditions pertaining to Scheme 15: (a) Me2NH, 40 C, 71 h, 61%.
119
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
Scheme 16:
0 H O
r H O
00 OMe a
0 OMe b
_...
N/ 00 - 0 N/ O. i 0
b b
Fi ñ
402-49 33
HOõ HO.,
r H O r
00 OMe c H O00 OMe
NC NC es i 0
HO I:I 0 Fl
34 63295
Reagents and conditions applicable to Scheme 16 are: (a) NH2OH-HC1, Na0Ac,
Et0H, H20, 80 C, 27 h, 72%; (b) Na0Me, Me0H, 55 C, 1 h; (c) 0 1,3-dibromo-
5,5-
dimethylhydantoin, 0 C, 40 min; (ii) pyridine, 55 C, 7 h, 27% from 33.
Scheme 17:
HO., 0
jj H 0
4
OMe a NH H 4110 1)At
_,,..
iip OMe b
_,..
N/ O. o
NI I e. ; 0
O 0
A n
33 35
0 0
NH H 411i NH H 410
c
_...
NC tes Ali 0 OMe NC OMe
HO 0 O. i 0
H H
36 63296
Reagents and conditions applicable to Scheme 17 are: (a) POC13, pyridine, rt,
5 h,
75%; (b) Na0Me, Me0H, 55 C, 4 h, 93% (i) 1,3-dibromo-5,5-dimethylhydantoin, 0
C, 1 h;
(ii) pyridine, 55 C, 23 h, 93%.
120
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
Scheme 18:
?
O e o
o 0
00 CO2Me 0 CO2Me
N as/ le. E N/ 00 -
0 b
. _
---,H .----1:1
402-49 37
.
0
0 O 0
0 O
,
0 CO2Me 0 CO2Me
b NCle-. c NC
,
1*-0
----1=1 ----1=1
38 63263
Reagents and conditions applicable to Scheme 18 are: (a) m-CPBA, rt, 48 h,
22%; (b)
Na0Me, 55 C, 2 h, 66%; (c) (i) DBDMH, 0 C, 1 h; (ii) pyridine, 55 C, 3 h,
72%.
Scheme 19:
0 HO
0 O 0 0
0 CO2Me a 0 CO2Me
N,/ le. - N,/ le. E
0 0
---H ----,1:1
,
39
37
HO___ 0 O 0 O
.0 c02me
.40 CO2Me
b NC c NC
õ.
(0.0 E õ.
0.0
HO .
----I:I ---fl
40 63289
Reagents and conditions pertaining applicable to Scheme 19 are: (a) LiA1H4, 0
C, 40
min, 62%; (b) Na0Me, 55 C, 2 h, 83%; (c) (i) DBDMH, 0 C, 1 h; (ii) pyridine,
55 C, 4 h,
40%.
Example 3 ¨ Synthesis and Characterization of Oleanolic Acid Derivatives
Compound 2: Bleach (5.25 wt% of NaC10 (aq), 129 mL, 91 mmol) was added to a
stirred solution of compound 1 (34.67 g, 71 mmol) in AcOH (471 mL) at room
temperature.
121
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
After stirring for 40 min, the reaction mixture was poured into ice-water (1.5
L) and stirred
for 5 min. The white precipitate was collected by filtration and washed
thoroughly with
water. The filtered solid was then dissolved in Et0Ac and washed with NaHCO3
(aq)
solution, dried with MgSO4 and concentrated. The residue obtained was purified
by column
chromatography (silica gel, 10% to 25% Et0Ac in hexanes) to give product 2
(27.8 g, 80%)
as a white foam solid: 1H NMR (400 MHz, CDC13) 6 3.69 (s, 3H), 2.80 (m, 1H),
2.65 (d, 1H,
J= 4.0 Hz), 2.53 (ddd, 1H, J= 7.2, 10.8, 16.0 Hz), 2.38 (ddd, 1H, J = 3.6,
6.8, 16.0 Hz),
2.16-2.30 (m, 2 H), 1.95 (m, 1H), 1.89 (m, 1H), 1.80 (m, 2H), 1.62-1.73 (m,
3H), 1.57 (m,
2H), 1.47 (m, 2H), 1.15-1.40 (m, 7H), 1.09 (s, 3H), 1.05 (s, 3H), 1.01 (s,
3H), 0.99 (s, 3H),
0.98 (s, 3H), 0.95 (s, 3H), 0.90 (s, 3H); m/z 485.3 (M+1).
Compound 402-48: Na0Me solution (25% w/w in Me0H, 132.3 mL, 570 mmol) was
added to a solution of compound 2 (27.6 g, 57 mmol) in Me0H (250 mL) under
nitrogen.
The reaction mixture was heated to 55 C in an oil bath, and HCO2Et (93 mL,
1.15 mmol, 20
eq) was added dropwise via an addition funnel. The reaction mixture was
stirred at 55 C for
24 h and then at room temperature for another 40 h. After removal of Me0H (150
mL) by
evaporation, t-BuOMe (200 mL) was added, and the mixture was cooled to 0 C.
12 N HC1
(aq) (50 mL, 600 mmol, 10.5 eq) was then added over 10 min, and the mixture
was extracted
with Et0Ac. The combined extracts were washed with water, dried with MgSO4,
and
concentrated. The brown oil obtained was purified by column chromatography
(silica gel,
5% to 10% Et0Ac in hexanes) to give product 402-48 (20.5 g, 70%) as a white
foam solid:
1H NMR (400 MHz, CDC13) 6 14.89 (d, 1H, J= 3.2 Hz), 8.61 (d, 1H, J= 3.2 Hz),
3.69 (s,
3H), 2.80 (m, 1H), 2.67 (d, 1H, J= 4.0 Hz), 2.20-2.34 (m, 3H), 1.98 (m, 1H),
1.62-1.92 (m,
6H), 1.10-1.56 (m, 10H), 1.20 (s, 3H), 1.12 (s, 3H), 1.02 (s, 3H), 0.99 (s,
3H), 0.96 (s, 3H),
0.91 (s, 3H), 0.85 (s, 3H); m/z 513.3 (M+1).
Compound 402-49: A mixture of compound 402-48 (20.3 g, 40 mmol) and
NH2OH=HC1 (4.12 g, 59 mmol) in Et0H (300 mL) and water (60 mL) was heated at
55 C for
14 h. After cooling to room temperature, Et0H was removed by evaporation, and
the white
slurry obtained was extracted with Et0Ac. The combined extracts were washed
with water,
dried with MgSO4, and concentrated. The residue obtained was purified by
column
chromatography (silica gel, 10% to 20% Et0Ac in hexanes) to give product 402-
49 (18.8 g,
93%) as a white solid: 1H NMR (400 MHz, CDC13) 6 7.99 (s, 1H), 3.70 (s, 3H),
2.81 (m, 1H),
2.68 (d, 1H, J= 4.4 Hz), 2.37 (d, 1H, J= 15.2 Hz), 2.23-2.33 (m, 2H), 1.76-
1.98 (m, 5H),
122
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
1.68 (m, 3H), 1.11-1.62 (m, 9H), 1.32 (s, 3H), 1.23 (s, 3H), 1.02 (s, 3H),
0.99 (s, 3H), 0.97 (s,
3H), 0.91 (s, 3H), 0.84 (s, 3H); m/z 510.3 (M+1).
Compound 402-46: Na0Me (25% w/w in Me0H, 8.75 mL, 38 mmol) was added
dropwise to a suspension of 402-49 (16.16 g, 31.7 mmol) in Me0H (55 mL) at 0
C under N2.
The reaction mixture was heated at 55 C for 2 h and then cooled to 0 C. t-
BuOMe (150 mL)
and 1 N HC1 (aq) (50 mL) were added successively, and the mixture was
extracted with
Et0Ac. The combined extracts were washed with water, dried with MgSO4, and
concentrated to give compound 402-46 (17.80 g, 100%) as a white foam solid.
402-46 is a
mixture of two equilibrium forms, the enol form (as shown in Scheme 1) and the
ketone form,
in the ratio of 2:3. 1H NMR of the mixture: (400 MHz, CDC13) 6 5.69 (s, 0.4H),
3.87 (m,
0.6H), 2.80 (m, 1H), 2.65 (m, 1H), 0.82-2.30 (m, 44H); m/z 510.3 (M+1).
Compound 402-02: 1,3-Dibromo-5,5-dimethylhydantoin (5.98 g, 20.9 mmol) was
added to a solution of compound 402-46 (17.76 g, 35 mmol) in DMF (75 mL) at 10
C. After
stirring at room temperature for 2 h, pyridine (8.5 mL, 105 mmol) was added,
and the
reaction mixture was heated at 55 C for 15 h. After cooling to room
temperature, the
mixture was poured into water (700 mL) and stirred for 5 min. The pale brown
precipitate
was collected by filtration and washed with water. The solid was dissolved in
CH2C12, and
the solution was washed with 1 N HC1 (aq) and water, then dried with MgSO4 and
concentrated. The residue obtained was purified by column chromatography
(silica gel, 0%
to 70% Et0Ac in hexanes) to give product 402-02 (14.3 g, 81%) as a white
solid: 1H NMR
(400 MHz, CDC13) 6 7.65 (s, 1H), 3.69 (s, 3H), 2.82 (m, 1H), 2.68 (d, 1H, J =
4.4 Hz), 2.44
(dd, 1H, J= 4.8, 16.0 Hz), 2.35 (dd, 1H, J= 12.8, 16.0 Hz), 1.86-2.00 (m, 3H),
1.81 (m, 1H),
1.60-1.71 (m, 4H), 1.42-1.55 (m, 3H), 1.24 (m, 1H), 1.10-1.24 (m, 4H), 1.22
(s, 3H), 1.16 (s,
3H), 1.15 (s, 3H), 1.07 (s, 3H), 0.99 (s, 3H), 0.97 (s, 3H), 0.92 (s, 3H); m/z
508.2 (M+1).
Compound 402-51: A stream of nitrogen was bubbled through a stirring solution
of
compound 402-02 (6.31 g, 12.4 mmol) and LiI (33.35 g, 248 mmol) in DMF (87 mL)
at 160
C for 8 h. After cooling to 50 C, the reaction mixture was diluted with Et0Ac
(100 mL). 1
N HC1 (aq) solution (30 mL) was then added at room temperature and stirred for
5 min. The
mixture was extracted with Et0Ac, and the combined extracts were washed with
water, 10%
Na25203 (aq) and water, then dried with Mg504 and concentrated. The residue
obtained was
purified by column chromatography (silica gel, 5% to 50% Et0Ac in CH2C12) to
give acid
402-51 (6.02 g, 95%) as a white solid: 1H NMR (400 MHz, CDC13) 6 10.41 (bs,
1H), 7.65 (s,
1H), 2.80 (m, 1H), 2.74 (d, 1H, J= 4.4 Hz), 2.46 (dd, 1H, J= 4.8, 16.0 Hz),
2.37 (dd, 1H, J =
123
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
12.8, 16.0 Hz), 1.86-2.02 (m, 4H), 1.44-1.79 (m, 8H), 1.35 (m, 1H), 1.12-1.29
(m, 3H), 1.22
(s, 3H), 1.16 (s, 3H), 1.14 (s, 3H), 1.11 (s, 3H), 0.99 (s, 3H), 0.98 (s, 3H),
0.93 (s, 3H); m/z
494.3 (M+1).
Compound 402-64: NaHCO3 (78 mg, 0.93 mmol) and Dess-Martin periodinane (99
mg, 0.23 mmol) were added successively to a solution of 402-63 (45 mg, 94
gmol) in CH2C12
(5 mL) at room temperature. After stirring for 1 h, 5% Na2S203 (aq) solution
was added. The
reaction mixture was extracted with t-BuOMe, and the combined extracts were
washed with
NaHCO3 (aq) solution, dried with MgSO4, and concentrated. The crude product
obtained was
purified by column chromatography (silica gel, 0% to 35% Et0Ac in hexanes) to
give 402-64
(22 mg, 49%) as a white foam solid: 1H NMR (300 MHz, CDC13) 6 9.33 (s, 1H),
7.62 (s, 1H),
2.61 (m, 1H), 2.50 (d, 1H, J= 4.4 Hz), 2.45 (dd, 1H, J= 4.8, 16.4 Hz), 2.34
(dd, 1H, J =13.2,
16.4 Hz), 1.92-2.00 (m, 2H), 1.88 (m, 1H), 1.42-1.74 (m, 9 H), 1.28-1.35 (m,
2H), 1.21 (s,
3H), 1.20 (m, 1H), 1.15 (s, 3H), 1.14 (s, 3H), 1.12 (m, 1H), 1.06 (s, 3H),
0.97 (s, 6H), 0.93 (s,
3H); m/z 478.2 (M+1).
Compound 402-59: To a solution of 402-51 (2.08 g, 4.21 mmol) in CH2C12 (28 mL)
were successively added oxalyl chloride (1.07 mL, 12.64 mmol) and DMF (5
drops, cat.) at 0
C. The reaction was allowed to warm to room temperature and was stirred for 3
h. The
reaction mixture was concentrated and dried in vacuo 30 min, to give acid
chloride 3 as a
yellow solid, which was used directly in the next step. To a solution of 3
(2.16 g, 4.21 mmol)
in THF (28 mL) at 0 C was added ammonia (2.0 M solution in Me0H, 11 mL, 22.00
mmol).
The reaction was allowed to warm to room temperature and was stirred for 5 h.
The solvents
were then evaporated, and the residue was extracted with Et0Ac. The extracts
were washed
with water, 1 N HC1 (aq), and water, then dried over MgSO4, filtered, and
concentrated to
give 402-59 (2.06 g, 99%) as a pale yellow solid. A small amount (53 mg) was
purified by
column chromatography (silica gel, 0% to 25% Et0Ac in CH2C12) to give higher
purity 402-
59 (14 mg, white solid) for biological assay: 1H NMR (400 MHz, CDC13) 6 7.65
(s, 1H), 5.63
(br s, 1H), 5.36 (br s, 1H), 2.90 (br d, 1H, J= 5.2 Hz), 2.71 (br d, 1H, J= 12
Hz), 2.42 (m,
2H), 1.96-2.10 (m, 4H), 1.78-1.90 (m, 2H), 1.45-1.69 (m, 6H), 1.23-1.40 (m,
4H), 1.22 (s,
3H), 1.16 (s, 3H), 1.15 (s, 3H), 1.13 (s, 3H), 0.99 (s, 3H), 0.98 (s, 3H),
0.93 (s, 3H); m/z
493.3 (M+1)
Compound 402-57: A solution of 402-59 (2.01 g, 4.08 mmol) in CH2C12 (28 mL)
was
prepared and cooled to 0 C. To this solution were added TFAA (0.91 mL, 6.55
mmol) and
Et3N (1.48 mL, 10.62 mmol). The reaction was stirred at 0 C for 3 h, after
which it was
124
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
quenched by the addition of saturated NaHCO3 (aq) solution (40 mL). After
stirring for 10
min, the reaction mixture was extracted with CH2C12 and washed with saturated
NaHCO3
(aq), water, 1 N HC1 (aq), and water. The extracts were dried over MgSO4,
filtered, and
concentrated. The crude product was purified by column chromatography (silica
gel, 5% to
35% Et0Ac in hexanes). The purified product was triturated with Et0H, then
filtered and
dried on the filter to give 402-57 (0.87 g, 45%) as a powdery white solid: 1H
NMR (400
MHz, CDC13) 6 7.63 (s, 1H), 3.04 (d, 1H, J= 4.4 Hz), 2.38-2.57 (m, 3H), 1.91-
2.19 (m, 5H),
1.61-1.78 (m, 4H), 1.44-1.54 (m, 3H), 1.32 (s, 3H), 1.26-1.30 (m, 4H), 1.22
(s, 3H), 1.19 (s,
3H), 1.16 (s, 3H), 0.99 (s, 3H), 0.95 (s, 3H), 0.92 (s, 3H); m/z 475.2 (M+1).
Compound 404-02: To a solution of 3 (3.03 g, 5.92 mmol) in toluene (84 mL) was
added NaHCO3 (1.98 g). A solution of trifluoroethylamine hydrochloride (5.64
g, 41.62
mmol) in water (14 mL) was prepared, then added to the reaction. The reaction
was heated to
70 C and stirred for 2 h. After cooling to room temperature, the reaction
mixture was
extracted with Et0Ac and washed with brine. The combined extracts were dried
over
MgSO4, filtered, and concentrated.
The crude product was purified by column
chromatography (silica gel, 0% to 40% Et0Ac in CH2C12) to give 404-02 (2.35 g,
69%) as a
white solid: 1H NMR (400 MHz, CDC13) 6 7.65 (s, 1H), 5.94 (br t, 1H, J= 8 Hz),
4.10 (m,
1H), 3.69-3.88 (m, 1H), 2.84 (d, 1H, J= 8 Hz), 2.78 (br d, 1H, J= 16 Hz), 2.38
(m, 2H), 2.12
(m, 1H), 2.06 (m, 2H), 1.61-1.83 (m, 5H), 1.24-1.52 (m, 8H), 1.22 (s, 3H),
1.16 (s, 3H), 1.14
(s, 3H), 1.07 (s, 3H), 0.99 (s, 6H), 0.93 (s, 3H); m/z 575.3 (M+1).
Compounds 4, 5: To a solution of 402-49 (395 mg, 0.775 mmol) in THF (7.8 mL)
was added LiA1H4 (1.0 M solution in THF, 0.78 mL, 0.780 mmol) at 0 C. The
reaction was
stirred at 0 C for 40 min, after which it was quenched by the addition of
water (5 mL) and
stirred 5 min. The reaction mixture was extracted with Et0Ac and washed with
water. Solid
NaC1 was added to break up emulsions. The combined extracts were dried over
Na2504,
filtered, and concentrated. The crude product was purified by column
chromatography (silica
gel, 10% to 70% Et0Ac in hexanes) to give both 4 (151 mg, 38%) as a white
solid and to
give 5 (134 mg, 34%) as a white solid:
Compound 4: 1H NMR (400 MHz, CDC13) 6 7.99 (s, 1H), 3.79 (m, 1H), 3.72 (s,
3H), 2.75
(m, 1H), 2.52 (d, 1H, J= 14.4 Hz), 1.95-2.11 (m, 2H), 1.57-1.88 (m, yyH), 1.24-
1.54 (m,
yyH), 1.30 (s, 3H), 1.20 (s, 3H), 1.00 (s, 3H), 0.93 (s, 6H), 0.92 (s, 3H),
0.82 (s, 3H); m/z
512.3 (M+1).
Compound 5: m/z 494.3 (M-17), 434.3 (M-17-60).
125
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
Compound 6: To a solution of 4 (371 mg, 66 nmol) in Me0H (7.3 mL) was added
Na0Me (25 wt % solution in Me0H, 0.42 mL, 1.837 mmol) at room temperature. The
reaction was heated to 55 C and stirred 7 h. After cooling the reaction to
room temperature,
the reaction mixture was diluted with MTBE (10 mL), then quenched with 1 N HC1
(aq) (10
mL). The reaction mixture was extracted with Et0Ac and washed with 1 N HC1
(aq) and
brine. The combined extracts were dried over Na2SO4, filtered, and
concentrated to give 6
(361 mg, 94%) as a white solid. Compound 6 is a mixture of two equilibrium
forms, the enol
form (as shown in Scheme 5) and the ketone form, in the ratio of 2:3. 1H NMR
of the
mixture: (400 MHz, CDC13) 6 5.66 (d, 0.4H, J= 4.8 Hz), 4.09 (br, 1H), 3.90 (m,
0.6H), 3.71
(s, 1.2H), 3.68 (s, 1.8H), 2.73 (m, 1H), 2.48 (m, 1H), 2.14-2.26 (m, 2H), 0.80-
2.02 (m, 39H);
m/z 494.3 (M-17), 434.3 (M-77).
Compound 402-66: A solution of 6 (361 mg, 0.705 mmol) in DMF (7.1 mL) was
prepared. 1,3-Dibromo-5,5-dimethylhydantoin (120 mg, 0.420 mmol) was added,
and the
reaction was stirred at room temperature for 1 h. Pyridine (0.23 mL, 2.858
mmol) was added,
and the reaction was heated to 55 C and stirred 10 h. After cooling the
reaction to room
temperature, the reaction mixture was extracted with Et0Ac and washed with 5%
Na2S203
(aq), water, 1 N HC1 (aq), and water. The Et0Ac extracts were dried over
Na2504, filtered,
and evaporated. The crude product was purified by column chromatography
(silica gel, 5%
to 40% Et0Ac in hexanes) to give 402-66 (127 mg, 35% yield) as a white solid;
1H NMR
(400 MHz, CDC13) 6 7.81 (s, 1H), 3.81 (ddd, 1H, J= 4.8, 10.8, 15.6 Hz), 3.72
(s, 3H), 2.75
(m, 1H), 2.01 (m, 2H), 1.74-1.88 (m, 3H), 1.24-1.72 (m, 15 H), 1.19 (s, 3H),
1.13 (s, 3H),
1.12 (s, 3H), 0.98 (s, 3H), 0.96 (s, 3H), 0.95 (s, 3H), 0.94 (s, 3H); m/z
492.3 (M-17), 432.3
(M-77).
Compound 7: Using the procedure described for the synthesis of compound 6 from
compound 4, compound 7 (96 mg, 89% yield) was produced from compound 5 (108
mg,
0.211 mmol): m/z 494.3 (M-17).
Compound 63219: Using the procedure described for the synthesis of compound
402-66 from compound 6, compound 63219 (30 mg, 32% yield) was produced from
compound 7 (95 mg, 0.186 mmol): 1H NMR (400 MHz, CDC13) 6 7.81 (1H, s), 4.16
(1H, bs),
3.68 (3H, s), 2.44-2.54 (2H, m), 1.98-2.10 (2H, m), 1.78-1.94 (4H, m), 1.42-
1.76 (7H, m),
1.00-1.42 (6H, m), 1.32 (3H, s), 1.23 (3H, s), 1.13 (3H, s), 1.11 (3H, s),
0.94 (3H, s), 0.93
(3H, s), 0.92 (3H, s); m/z 492.3 (M-17).
126
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
Compound 8: Oxalyl chloride (0.11 mL, 1.30 mmol) and catalytic amount of DMF
were added sequentially to a solution of compound 402-51 (200 mg, 0.41 mmol)
in CH2C12 (4
mL) at 0 C. The reaction mixture was warmed to room temperature and stirred
for 2 h.
After removing the solvent by evaporation, the crude acid chloride was
obtained as a light
yellow foam solid. Hydrazine hydrate (64% of hydrazine, 0.50 mL) was added to
a solution
of acid chloride in Et20 (8 mL) at 0 C. After stirring for 30 min, CH2C12 was
added. The
mixture was washed with water, dried over MgSO4, filtered and evaporated to
give compound
8 (200 mg, 97% yield) as white solid, which was used in the next step without
further
purification: m/z 508.3 (M+1).
Compound 9: Et3N (0.12 mL, 0.86 mmol) and acetyl chloride (37 ilL, 0.52 mmol)
were added sequentially to a solution of compound 8 (200 mg, 0.39 mmol) in
CH2C12 (4 mL)
at r.t. After stirring for 30 min, Et3N (0.36 mL, 2.59 mmol) and acetyl
chloride (110 ilL, 1.55
mmol) were added again. After stirring for another 30 min, NaHCO3 (aq.)
solution was
added to quench the reaction. The reaction mixture was transferred to a
separatory funnel,
and extracted with Et0Ac. The combined extracts were washed with water, dried
over
MgSO4, filtered and evaporated. The residue was purified by silica gel
chromatography (0%
to 75% Et0Ac in hexanes) to give compound 9 (180 mg, 77% yield) as a white
foam solid:
m/z 592.3 (M+1).
Compound 63264: Na0Me (25% w/w in Me0H, 0.14 mL, 0.61 mmol) was added to
a solution of compound 9 (180 mg, 0.30 mmol) in Me0H (3 mL) at 0 C. After
stirring at r.t.
for 10 min, the reaction mixture was treated with t-BuOMe (10 mL) and 1N HC1
(aq.) (1
mL), which was then transferred to a separatory funnel and extracted with
Et0Ac. The
combined extracts were washed with NaHCO3 (aq.) solution, dried over MgSO4,
filtered and
evaporated. The residue was purified by silica gel chromatography (0% to 100%
Et0Ac in
hexanes) to give compound 63264 (121 mg, 72% yield) as a white solid: 1H NMR
(400 MHz,
CDC13) 6 7.98 (d, 1H, J= 4.4 Hz), 7.77 (d, 1H, J= 4.4 Hz), 7.65 (s, 1H), 2.89
(d, 1H, J= 4.4
Hz), 2.82 (m, 1H), 2.34-2.45 (m, 2H), 2.10 (m, 1H), 2.08 (s, 3H), 1.82-2.02
(m, 4H), 1.60-
1.69 (m, 3H), 1.44-1.53 (m, 4H), 1.16-1.40 (m, 4H), 1.22 (s, 3H), 1.16 (s,
3H), 1.15 (s, 3H),
1.13 (s, 3H), 0.99 (s, 3H), 0.98 (s, 3H), 0.94 (s, 3H); m/z 550.3 (M+1).
Compound 63267: A solution of compound 63264 (74 mg, 0.13 mmol), Ts0H (13
mg, 0.068 mmol) in toluene (5 mL) was heated at reflux with dean-stark trap
for 1 h. After
cooling to r.t., the reaction mixture was transferred to a separatory funnel,
washed with
NaHCO3 (aq.) solution, dried over Mg504, filtered and evaporated. The residue
was purified
127
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
by silica gel chromatography (0% to 100% Et0Ac in hexanes) to give compound
63267 (24
mg, 33% yield) as a white foam solid: 1H NMR (400 MHz, CDC13) 6 7.64 (s, 1H),
2.94 (m,
1H), 2.79 (d, 1H, J= 4.4 Hz), 2.54 (s, 3H), 2.46 (m, 1H), 2.34 (m, 1H), 2.21
(m, 1H), 1.84-
2.06 (m, 5H), 1.56-1.70 (m, 4H), 1.24-1.47 (m, 5H), 1.23 (s, 3H), 1.18 (m,
1H), 1.15 (s, 3H),
1.13 (s, 3H), 1.05 (s, 3H), 1.02 (s, 3H), 0.98 (s, 3H), 0.93 (s, 3H); m/z
532.3 (M+1).
Compound 11: Et3N (1.46 mL, 10.49 mmol) and TFAA (0.88 mL, 6.33 mmol) were
added sequentially to a solution of compound 10 (1.97 g, 4.20 mmol) in CH2C12
(42 mL) at 0
C. After stirring for 1.5 h, NaHCO3 (aq.) solution was added to the reaction
mixture, which
was then transferred to a separatory funnel and extracted with CH2C12. The
combined
extracts were dried over MgSO4, filtered and evaporated. The residue was
purified by silica
gel chromatography (0% to 35% Et0Ac in hexanes) to give compound 11 (1.62 g,
85% yield)
as a white solid: m/z 452.3.
Compound 12: A solution of Bu3SnN3 (1.00 mL, 3.62 mmol) and compound 11 (1.36
g, 3.02 mmol) in xylene (5.0 mL) was heated at reflux for 48 h. After cooling
to r.t., the
reaction mixture was purified by silica gel chromatography (0% to 30% Et0Ac in
CH2C12) to
give compound 12 (994 mg, 67% yield) as a light yellow foam solid: m/z 493.3
(M+1).
Compound 13: Na0Me solution (25% w/w in Me0H, 1.16 mL, 5.07 mmol) was
added dropwise to a mixture of compound 12 (168 mg, 0.34 mmol) and HCO2Et
(0.82 mL,
10.19 mmol) at 0 C under N2. After stirring at room temperature for 1 h, t-
BuOMe (10 mL)
was added. The mixture was cooled to 0 C, and 12 N HC1 (aq) (0.42 mL, 5.04
mmol) was
added slowly. The mixture was transferred to a separatory funnel and extracted
with Et0Ac.
The combined extracts were washed with water, dried over MgSO4 and
concentrated to give
crude 2-formyl ketone, which was then mixed with NH2OH=HC1 (36 mg, 0.51 mmol),
Et0H
(4 mL), and water (0.4 mL), and heated at 60 C for 3 h. After removing Et0H
by
evaporation, the white slurry obtained was transferred to a separatory funnel
and extracted
with CH2C12. The combined extracts were washed with water, dried over Mg504,
and
concentrated. The residue was purified by column chromatography (silica gel,
0% to 30%
Et0Ac in CH2C12) to give compound 13 (95 mg, 54% yield) as a white foam solid:
m/z 520.3
(M+1).
Compound 63229: Using the procedure described for the synthesis of compound
402-
66 from compound 4, 63229 (12 mg, 60% yield) was produced from compound 13 (20
mg,
0.038 mmol): 1H NMR (400 MHz, CDC13) 6 7.70 (s, 1H), 3.03 (m, 1H), 2.69 (d,
1H, J= 4.0
Hz), 2.52 (dd, 1H, J= 4.4, 16.8 Hz), 2.29-2.36 (m, 2H), 1.96-2.03 (m, 3H),
1.56-1.82 (m,
128
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
6H), 1.25-1.57 (m, 6H), 1.22 (s, 3H), 1.18 (m, 1H), 1.13 (s, 3H), 1.11 (s,
3H), 1.04 (s, 3H),
1.03 (s, 3H), 0.98 (s, 3H), 0.75 (s, 3H); m/z 518.3 (M+1).
Compound 14: TMSCHN2 (2.0 M in Et20, 89 L, 0.18 mmol) was added to a
solution of compound 13 (84 mg, 0.16 mmol) in THF (1.25 mL) and Me0H (0.31 mL)
at 0
C. After stirring at room temperature for 10 min, acetic acid was added to
quench the
reaction. The reaction mixture was diluted with Et0Ac, transferred to a
separatory funnel,
washed with NaHCO3 (aq.) solution, dried over MgSO4 and evaporated. The
residue was
purified by column chromatography (silica gel, 0% to 60% Et0Ac in hexanes) to
give
compound 14 (67 mg, 77% yield) as a white solid: m/z 534.3 (M+1).
Compound 63230: Using the procedure described for the synthesis of compound
402-
66 from compound 4, 63230 (45 mg, 69% yield) was produced from compound 14 (65
mg,
0.12 mmol): 1H NMR (400 MHz, CDC13) 6 7.62 (s, 1H), 4.32 (s, 3H), 3.11 (m,
1H), 2.68 (d,
1H, J= 4.4 Hz), 2.42 (dd, 1H, J= 4.8, 16.4 Hz), 2.27 (dd, 1H, J = 13.2, 16.4
Hz), 2.22 (dd,
1H, J= 4.4, 14.8 Hz), 1.94-2.04 (m, 3H), 1.79 (m, 1H), 1.54-1.63 (m, 5H), 1.36-
1.50 (m, 4H),
1.26 (m, 1H), 1.20 (s, 3H), 1.13 (m, 1H), 1.12 (s, 3H), 1.09 (s, 3H), 1.05 (s,
3H), 1.01 (s, 3H),
0.97 (s, 3H), 0.70 (s, 3H); m/z 532.3 (M+1).
Compound 63223: Using the procedure described for the synthesis of compound 13
from compound 12, compound 63223 (1.95 g, 47% yield) was produced from
compound 15
(3.93 g, 7.12 mmol) as a pale yellow solid: 1H NMR (400 MHz, CDC13) 6 7.98
(1H, s), 5.91
(1H, t, J = 6.0 Hz), 4.00-4.15 (1H, m), 3.55-3.90 (1H, m), 2.72-2.82 (2H, m),
2.20-2.40 (3H,
m), 1.88-2.16 (4H, m), 1.10-1.84 (13H, m), 1.31 (3H, s), 1.21 (3H, s), 1.00
(3H, s), 0.98 (6H,
s), 0.91 (3H, s), 0.82 (3H, s); m/z 577.3 (M+H).
Compound 63227: Using the procedure described for the synthesis of compound 6
from compound 4, compound 63227 (1.64 g, quantitative yield) was produced from
compound 63223 (1.61 g, 2.79 mmol): 1H NMR (400 MHz, CDC13) for enol form: 6
5.91
(1H, t, J = 6.0 Hz), 5.78 (1H,bs, enol), 4.00-4.16 (1H, m), 3.75-3.94 (1H, m),
2.70-2.85 (2H,
m), 1.90-2.30 (5H, m), 0.80-1.88 (36H, m); m/z 577.3 (M+H) (for both enol and
ketone
isomers).
Compound 63237: A solution of 63227 (1.61 g, 2.79 mmol) in DMF (9.3 mL) was
prepared. 1,3-Dibromo-5,5-dimethylhydantoin (456 mg, 1.59 mmol) was added, and
the
reaction was stirred at room temperature for 3 h. Pyridine (0.67 mL, 8.33
mmol) was added,
and the reaction was heated to 55 C and stirred 16 h. After cooling the
reaction to room
temperature, the reaction mixture was extracted with Et0Ac and washed with 5%
Na2S203
129
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
(aq), 1 N HC1 (aq), and water. The Et0Ac extracts were dried over Na2SO4,
filtered, and
evaporated. Compound 63237 was a minor component (18%) by crude LC-MS
analysis.
The crude product was purified by column chromatography (silica gel, 5% to 35%
Et0Ac in
hexanes) to give 63237 (188 mg, 12%) as a yellow foam solid: 1H NMR (400 MHz,
CDC13)
6 7.67 (s, 1H), 3.82 (m, 2H), 2.91 (m, 1H), 2.54 (m, 1H), 2.41 (m, 1H), 2.08
(m, 1H), 1.83-
1.94 (m, 2H), 1.52-1.74 (m, 6H), 1.39 (s, 3H), 1.22 (s, 6H), 1.20-1.49 (m,
7H), 1.16 (s, 3H),
0.98 (s, 3H), 0.96 (s, 3H), 0.95 (s, 3H); m/z 573.3 (M+1).
Compound 16: Using the procedure described for the synthesis of compound 4
from
compound 402-49, compound 16 (100 mg, 19% yield) was produced from compound
63223
(531 mg, 0.921 mmol): m/z 579.3 (M+1)
Compound 17: Using the procedure described for the synthesis of compound 6
from
compound 4, compound 17 (90 mg, 92% yield) was produced from compound 16 (98
mg,
0.169 mmol): m/z 561.3 (M-17).
Compound 63268: Using the procedure described for the synthesis of compound
402-66, compound 63268 (50 mg, 56% yield) was produced from compound 17 (90
mg,
0.156 mmol): 1H NMR (400 MHz, CDC13) 6 7.78 (s, 1H), 6.09 (t, 1H, J= 6.4 Hz),
4.06 (m,
1H), 3.89 (m, 1H), 3.78 (m, 1H), 2.70 (m, 1H), 1.98-2.05 (m, 2H), 1.84 (dd,
1H, J= 4.4, 10.8
Hz), 1.75 (ddd, 1H, J= 4.4, 13.6, 13.6 Hz), 1.20-1.62 (m, 15H), 1.18 (s, 3H),
1.11 (s, 3H),
1.10 (s, 3H), 1.06 (m, 1H), 0.98 (s, 3H), 0.95 (s, 3H), 0.94 (s, 3H), 0.91 (s,
3H); m/z 559.3
(M-17).
Compound 19: Na0Me (25 w/w% solution in Me0H, 7.29 mL, 31.88 mmol) was
added to a solution of compound 18 (1.00 g, 2.12 mmol) in ethyl formate (5.13
mL, 63.78
mmol) at 0 C. After stirring for 1.5 h, t-BuOMe (10 mL) and 12 N (aq.) HC1
(2.66 mL,
31.92 mmol) were added sequentially. After stirring for another 5 min, the
reaction mixture
was transferred to a separatory funnel, which was extracted with Et0Ac. The
combined
extracts were washed with water. The organic layer was separated, which was
dried over
MgSO4, filtered, and concentrated. The crude product was mixed with NH2OH-HC1
(0.22 g,
3.17 mmol), water (2 mL) and Et0H (35 mL). The reaction mixture was heated at
65 C for
3.5 h, after which Et0H was removed by evaporation. The residue was
partitioned between
Et0Ac and water. The organic extract was separated, which was dried over
MgSO4, filtered,
and evaporated. The residue was purified by silica gel chromatography (0% to
60% Et0Ac
in hexanes) to give compound 19 (820 mg, 78% yield) as a white solid: m/z
496.3 (M+1).
130
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
Compound 20: Oxalyl chloride (110 [iL, 1.30 mmol) was added to a solution of
compound 19 (195 mg, 0.39 mmol) in CH2C12 (4 mL) at 0 C, followed by the
addition of
catalytic amount of DMF. The reaction was stirred at room temperature for 2 h,
after which
CH2C12 was evaporated under vacuum to give acid chloride as a light yellow
foam solid.
Et3N (113 [iL, 0.81 mmol) and a solution of acethydrazide (50 mg, 0.67 mmol)
in CH2C12 (2
mL) were added sequentially to a suspension of the acid chloride in ether (4
mL) at 0 C. The
reaction was warmed to room temperature and stirred for 30 min. Et0Ac was then
added,
and the crude mixture was transferred to a separatory funnel, which was washed
with water,
1N (aq.) HC1, water. The organic layer was separated, which was dried over
MgSO4, filtered,
and concentrated. The crude product was purified by column chromatography
(silica gel, 0%
to 100% Et0Ac in hexanes) to give product 20 (215 mg, 99% yield) as a white
foam solid: 1H
NMR (400 MHz, CDC13) 6 8.14 (d, 1H, J = 5.2 Hz), 8.05 (d, 1H, J= 5.2 Hz), 8.00
(s, 1H),
2.86 (m, 2H), 2.34 (m, 3H), 2.09 (s, 3H), 1.80-2.18 (m, 8H), 1.34-1.74 (m,
7H), 1.33 (s, 3H),
1.23 (s, 3H), 1.16-1.26 (m, 2H), 1.08 (s, 3H), 1.00 (s, 6H), 0.93 (s, 3H),
0.84 (s, 3H).
Compound 21 and Compound 22: A suspension of compound 20 (215 mg, 0.39
mmol) and Lawesson's reagent (190 mg, 0.47 mmol) in toluene was heated at
reflux for 30
min. After cooling to room temperature, the reaction mixture was purified by
column
chromatography (silica gel, 0% to 65% Et0Ac in hexanes) to give product 21 (21
mg, 10%
yield) as a light yellow foam solid: m/z 550.3 (M+1). From the column,
compound 22 (60
mg, 29% yield) was also obtained as a white foam solid: m/z 534.3 (M+1).
Compound 23: Na0Me (25 w/w% solution in Me0H, 17 [iL, 0.074 mmol) was added
to a solution of compound 21 (33 mg, 0.060 mmol) in Me0H (0.6 mL) at room
temperature.
The reaction was then heated to 55 C, and stirred for 1 h. After cooling to 0
C, t-BuOMe
and 1 N (aq.) HC1 were added, and stirred for 5 min. The reaction mixture was
transferred to
a separatory funnel, which was extracted with Et0Ac. The combined Et0Ac
extracts were
washed with water, dried over MgSO4, filtered, and concentrated. The crude
product was
purified by column chromatography (silica gel, 0% to 45% Et0Ac in hexanes) to
give
product 23 (24 mg, 73% yield) as a white foam solid: m/z 550.3 (M+1). Compound
23 is an
isomeric mixture of ketone and enol forms.
Compound 63274: To a solution of compound 23 (23 mg, 0.041 mmol) in DMF (0.3
mL) was added 1,3-dibromo-5,5-dimethylhydantion (6.1 mg, 0.021 mmol) at 0 C,
and the
reaction was stirred at 0 C for 1 h. Pyridine (14 [LL, 0.17 mmol) was then
added, and the
mixture was heated at 55 C for 3 h. After cooling to room temperature, the
reaction was
diluted with Et0Ac, and transferred to a separatory funnel, which was then
washed with
131
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
Na2S03 (aq.) solution and water. The organic layer was separated, which was
dried over
MgSO4, filtered, and concentrated. The crude product was purified by
column
chromatography (silica gel, 0% to 45% Et0Ac in hexanes) to give product 63274
(18 mg,
79% yield) as a white foam solid: 1H NMR (400 MHz, CDC13) 6 7.61 (s, 1H), 2.97
(d, 1H, J
= 4.4 Hz), 2.89 (m, 1H), 2.74 (s, 3H), 2.42 (dd, 1H, J = 4.8, 16.4 Hz), 2.29
(dd, 1H, J= 13.6,
16.4 Hz), 2.29 (m, 1H), 2.02 (m, 1H), 1.94 (dd, 1H, J = 4.8, 13.2 Hz), 1.77-
1.91 (m, 3H),
1.52-1.66 (m, 4H), 1.36-1.50 (m, 4H), 1.26 (m, 1H), 1.19 (s, 3H), 1.13 (m,
1H), 1.11 (s, 3H),
1.09 (s, 3H), 1.02 (s, 3H), 1.00 (s, 3H), 0.96 (s, 3H), 0.80 (s, 3H); m/z
548.3 (M+1).
Compound 24: LiA1H4 solution (1.0 M in THF, 42 mL, 42 mmol) was added to a
solution of compound 1 (5.0 g, 10.3 mmol) in THF (100 mL) at room temperature
under N2.
After stirring for 20 min at room temperature, LiA1H4 solution (1.0 M in THF,
21 mL, 21
mmol) was added again and the reaction mixture was refluxed for 1 h. After
cooling to 0 C,
water (10 mL) was added dropwise, followed by the addition of 1N HC1 (aq) (300
mL). The
mixture was extracted with Et0Ac. The combined extracts were washed with
water, dried
with MgSO4, and concentrated. The residue obtained was mixed with CH2C12 (200
mL). The
white solid that precipitated was collected by filtration and washed with
CH2C12 (2x100mL)
to give compound 24 (500 mg, 10%) as a white solid. The combined filtrate was
loaded on a
silica gel column and eluted with 0% to 100% Et0Ac in hexanes to give
additional compound
24 (800 mg, 17%) as a white solid: 1H NMR (400 MHz, CDC13) 6 3.79 (m, 1H),
3.54 (m,
2H), 3.20 (dd, 1H, J= 4.8, 10.8 Hz), 1.98 (m, 1H), 1.12-1.88 (m, 23H), 1.03
(s, 3H), 0.98 (s,
6H), 0.91 (s, 3H), 0.86 (s, 3H), 0.85 (s, 3H), 0.77 (s, 3H), 0.65-1.10 (m,
3H); m/z 443.3 (M-
H20+1), 425.3 (100%, M-2xH20+1).
Compound 25: TEMPO (27 mg x 4, 0.17 mmol x 4) and IPh(OAc)2 (563 mg x 4,
1.74 mmol x 4) were added to a white slurry of compound 24 (725 mg, 1.59 mmol)
in CH2C12
(200 mL) and water (0.1 mL) at 0 h, 2 h, 24 h and 48 h at room temperature.
After stirring at
room temperature for 72 h (overall reaction time), the reaction mixture turned
into a clear
pink solution, which was then transferred to a separatory funnel and washed
with Na2503 (aq)
solution. The organic phase was separated, dried over Mg504, filtered, and
evaporated. The
residue was purified by silica gel chromatography (0% to 75% Et0Ac in hexanes)
to give
compound 25 (560 mg, 77%) as a white solid: 1H NMR (400 MHz, CDC13) 6 9.37 (d,
1H, J=
1.2 Hz), 3.77 (m, 1H), 3.18 (dd, 1H, J= 4.8, 11.2 Hz), 2.51 (m, 1H), 0.98-1.87
(m, 23H), 0.97
(s, 3H), 0.96 (s, 3H), 0.94 (s, 3H), 0.92 (m, 1H), 0.90 (s, 3H), 0.86 (s, 3H),
0.82 (s, 3H), 0.75
(s, 3H), 0.65 (m, 1H); m/z 441.3 (M-H20+1), 423.3 (M-2xH20+1).
132
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
Compound 26: To a stirred suspension of (Ph3PCH2C1)C1 (4.224 g, 12.1 mmol) in
THF (13 mL) was added a solution of n-BuLi (4.8 mL, 11.64 mmol, 2.5 M in
Hexanes)
dropwise within 5 minutes at 0 C, followed by the addition of HMPA (2.4 mL).
The reaction
was stirred at r.t. for 20 minutes and then Compound 25 (1.332 g, 2.90 mmol)
in THF (13.0
mL) was added within 1 minute. The reaction mixture was stirred at room
temperature for 2
h, then quenched with HC1 (1N, 20 mL) and extracted with Et0Ac (100 mL). The
organic
phase was washed by HC1 (1N, 10 mL), NaC1 (Sat., 20 mL), dried over Na2SO4,
filtered, and
concentrated. The crude product was purified by column chromatography (silica
gel, 0% to
10% to 30% Et0Ac in hexanes) to give compound 26 (1.2508 g, 87.8%, a mixture
of E/Z
isomers) as a white solid:
Compound 27: To a stirred solution of 26 (1.2508 g, 2.55 mmol) in THF (17 mL)
was added a solution of MeLi (5.16 mL, 15.44 mmol, 3 M in CH2(0E02) dropwise
within 1
minute at 0 C. The mixture was then stirred at room temperature for 28 h and
quenched with
HC1 (1N, 15 mL). The aqueous solution was extracted with Et0Ac (2 x 100 mL).
The
combined organic phase was washed with water, NaC1 (sat.), dried over Na2SO4,
filtered, and
concentrated to give compound 27 (1.0630 g, 91.7%) as a white solid: m/z 437.3
(M-OH).
Compound 28: To a stirred mixture of 27 (881.7 mg, 1.94 mmol), Na0Ac (628.6
mg, 4 eq.) in CH2C12 (40 mL) was added PCC (1.257 g, 3 eq.) in one-portion at
room
temperature. The mixture was then stirred at room temperature for 5 h and
diluted with a
solvent mixture of Et0Ac/Hexanes (1:1, 50 mL). The mixture was directly loaded
on a silica
gel pad, which was then eluted throughout with a solvent mixture of
Et0Ac/Hexanes (1:1).
The eluate was collected and concentrated to give a colorless crystalline
product. This crude
product was purified by column chromatography (silica gel, 0% to 10% to 25%
Et0Ac in
hexanes) to give compound 28 (685 mg, 78.8%) as a white solid: m/z 451.3
(M+1).
Compound 29: To a stirred suspension of 28 (22.0 mg, 0.0488 mmol) in HCO2Et
(0.118 mL, 1.46 mmol) was added a solution of Me0Na (0.167 mL, 0.732 mmol,
25%w/w in
Me0H) at 0 C. The mixture was then stirred at room temperature for 25 h,
diluted with
TBME (1.4 mL) and quenched with HC1 (0.126 mL, conc.) followed by water (3
mL). The
aqueous solution was extracted with Et0Ac (10 mL). The combined organic phase
was
washed with brine (5 mL), dried over Na2504, filtered, and concentrated to
give compound
29 as a light yellow foam, which was used directly in the next step.
Compound 30: Compound 29 was dissolved in EtOH (2.1 mL). To this solution
were added NH2ORHC1 (5.1 mg, 0.0732 mmol) and H20 (0.27 mL) at room
temperature.
The mixture was heated at 60 C for 18 h and then cooled to room temperature.
The organic
133
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
volatiles were removed in vacuo. The remaining mixture was extracted with
Et0Ac (10 mL).
The organic phase was washed with water, brine, dried over Na2SO4, filtered,
and
concentrated. The crude product was purified by column chromatography (silica
gel, 0% to
10% to 25% Et0Ac in hexanes) to give 30 (20.8 mg, 89.6% from 6) as a colorless
crystalline
solid: m/z 476.3 (M+1).
Compound 31: To a stirred suspension of 30 (20.8 mg, 0.0437 mmol) in a solvent
mixture of Me0H (0.66 mL) and THF (0.11 mL) was added a solution of Me0Na
(23.8 L,
0.105 mmol, 25%w/w in MeOH) at 55 C. The mixture was then stirred at 55 C
for 3 h,
cooled to room temperature and quenched with 1N HC1 (aq) (5 mL). The mixture
was
extracted by Et0Ac (15 mL). The organic phase was washed with brine, dried
over Na2504,
filtered, and concentrated to give compound 31 as a light yellow foam: m/z
476.3 (M-17).
Compound 63303: Compound 31 was dissolved in benzene (2 mL). To this solution
was added a solution of DDQ (10.4 mg, 0.0458 mmol) in benzene (1 mL) at 85 C.
The
mixture was stirred at 85 C for 1.5 h, cooled to room temperature, and
quenched with sat.
NaHCO3 (aq) (5 mL). The mixture was extracted with Et0Ac (30 mL). The organic
phase
was washed with sat. NaHCO3 (aq) and brine, then was dried over Na2SO4,
filtered, and
concentrated to give a solid residue (a mixture of starting material and
desired product),
which was then dissolved in pyridine (0.5 mL). To this solution were added
Ac20 (50 L)
and DMAP (cat.) at room temperature. The mixture was stirred at room
temperature for 30
min and then quenched with NaHCO3 (sat.). The mixture was extracted with Et0Ac
(20 mL).
The organic phase was washed with NaHCO3 (sat.), HC1 (1N), brine, dried over
Na2504,
filtered, and concentrated to give a crude mixture, which was purified by
column
chromatography (silica gel, 0% to 10% to 25% Et0Ac in hexanes) to give 63303
(8.1 mg,
39.1% from 30) as a colorless solid: 1H NMR (400 MHz, CDC13) 6 7.66 (s, 1H),
3.25 (d, 1H,
J= 4.0 Hz), 2.25-2.52 (m, 3H), 2.22 (s, 1H), 1.78-2.15 (m, 5H), 1.44-1.76 (m,
9H), 1.08-1.36
(m, 2H), 1.29 (s, 3H), 1.23 (s, 3H), 1.19 (s, 3H), 1.17 (s, 3H), 0.89 (s, 3H),
0.94 (s, 3H), 0.91
(s, 3H); m/z 474.3 (M+1).
Compound 32: Compound 31 (95 mg, 0.2 mmol) was dissolved in a solvent mixture
of acetone (3.5 mL) and water (1.5 mL). To this solution were added Hg504 (5.9
mg, 0.02
mmol) and H2504 (2 drops, conc.) at room temperature. The mixture was stirred
at 55 C for
20 h, cooled to room temperature, and quenched with water (20 mL) and 1 N
HC1(aq) (10
mL). The mixture was extracted with Et0Ac (30 mL). The organic phase was
washed with 1
N HC1(aq) , water, sat. NaHCO3 (aq), brine, dried over Na2504, filtered, and
concentrated to
give a white solid, which was purified by column chromatography (silica gel,
0% to 10% to
134
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
25% Et0Ac in hexanes) to give compound 32 (90.3 mg, 91.5%) as a white foam:
m/z 494.3
(M+1).
Compound 63308: The procedure described for the synthesis of product 63274
from
compound 23 was then employed to convert compound 32 into product TX63308
(36.1 mg,
73.4%) as a white foam: 1H NMR (400 MHz, CDC13) 6 7.65 (s, 1H), 2.75-2.85 (m,
1H), 2.67
(d, 1H, J = 4.4 Hz), 2.44 (dt, 1H, J = 16.4, 4.8 Hz), 2.35 (dt, 1H, J= 16.0,
13.2 Hz), 2.16 (s,
3H), 1.92-2.06 (m, 3H), 1.30-1.76 (m, 12H), 1.18-1.29 (m, 1H), 1.22 (s, 3H),
1.16 (s, 3H),
1.15 (s, 3H), 1.04 (s, 3H), 0.97 (s, 3H), 0.96 (s, 3H), 0.93 (s, 3H); m/z
492.3 (M+1).
Compound 63323: 1,8-Diazabicyclo[5,4,0]undec-7-ene (0.18 mL, 1.204 mmol) was
added to a suspension of compound 402-51 (402 mg, 0.814 mmol) in toluene (5.4
mL) at
room temperature. After stirring for 2 min, benzyl bromide (0.12 mL, 1.009
mmol) was
added. The reaction mixture was heated at 100 C for 6 h, after which it was
cooled to room
temperature. The reaction was then diluted with Et0Ac, and was transferred to
a separatory
funnel, which was washed with 1 N HC1(aq) and brine. The organic extracts were
separated,
which was dried over Na2SO4, filtered and concentrated. The crude product was
purified by
column chromatography (silica gel, 0% to 20% Et0Ac in hexanes) to give product
63323
(308 mg, 65% yield) as a pale yellow foam solid: 1H NMR (400 MHz, CDC13) 6
7.56 (s, 1H),
7.30-7.37 (m, 5H), 5.21 (d, 1H, J = 12.4 Hz), 5.07 (d, 1H, J= 12.4 Hz), 2.84
(m, 1H), 2.47 (d,
1H, J= 4.0 Hz), 2.36 (dd, 1H, J= 4.4, 16.0 Hz), 2.17 (dd, 1H, J = 13.6, 16.0
Hz), 1.81-1.92
(m, 4H), 1.20-1.72 (m, 12H), 1.19 (s, 3H), 1.12 (s, 3H), 1.07 (s, 3H), 0.99
(s, 3H), 0.90 (s,
6H), 0.65 (s, 3H); m/z 584.4 (M+1).
Compound 63325: MeONH2-HC1 (109 mg, 1.305 mmol), water (0.4 mL) and Et3N
(0.24 mL, 1.722 mmol) were added sequentially to a solution of compound 3 (439
mg, 0.857
mmol) in THF (4.2 mL) at room temperature. The reaction was then heated at 40
C for 4 h.
After cooling to room temperature, the reaction was diluted with Et0Ac, and
was transferred
to a separatory funnel, which was washed with 1 N HC1(aq) and brine. The
organic extracts
were separated, which was dried over Na2SO4, filtered and concentrated. The
crude product
was purified by column chromatography (silica gel, 10% to 75% Et0Ac in
hexanes) to give
product 63325 (165 mg, 37% yield) as a white solid: 1H NMR (400 MHz, CDC13) 6
8.37 (s,
1H), 7.63 (s, 1H), 3.76 (s, 3H), 2.86 (d, 1H, J= 4.0 Hz), 2.71 (m, 1H), 2.44
(dd, 1H, J = 4.8,
16.4 Hz), 2.34 (dd, 1H, J= 13.2, 16.4 Hz), 1.91-2.08 (m, 3H), 1.74-1.90 (m,
2H), 1.59-1.68
(m, 3H), 1.40-1.50 (m, 4H), 1.21 (s, 3H), 1.18-1.38 (m, 4H), 1.15 (s, 3H),
1.14 (s, 3H), 1.12
(s, 3H), 0.97 (s, 3H), 0.96 (s, 3H), 0.91 (s, 3H); m/z 523.4 (M+1).
135
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
Compound 63326: Me2NH (2.0 M solution in THF, 1.23 mL, 2.460 mmol) was
added to a solution of compound 3 (408 mg, 0.797 mmol) in THF (4.1 mL) at room
temperature. The reaction was then heated at 40 C for 71 h. After cooling to
room
temperature, the reaction was diluted with Et0Ac, and was transferred to a
separatory funnel,
which was washed with 1 N HC1(aq) and brine. The organic extracts were
separated, which
was dried over Na2SO4, filtered and concentrated. The crude product was
purified by column
chromatography (silica gel, 10% to 70% Et0Ac in hexanes) to give product 63326
(254 mg,
61% yield) as a white solid: 1H NMR (400 MHz, CDC13) 6 7.64 (s, 1H), 3.06 (s,
6H), 2.97
(m, 1H), 2.29-2.42 (m, 2H), 1.94-2.06 (m, 3H), 1.74-1.85 (m, 2H), 1.61-1.67
(m, 5H), 1.24-
1.54 (m, 6H), 1.20 (s, 3H), 1.14 (s, 3H), 1.13 (s, 3H), 1.10 (m, 1H), 1.05 (s,
3H), 0.99 (s, 3H),
0.96 (s, 3H), 0.91 (s, 3H); m/z 521.4 (M+1).
Compound 33: NH2OH-HC1 (705 mg, 10.145 mmol), Na0Ac (1.169 mg, 14.251
mmol) and water (3.3 mL) were added to a suspension of compound 402-49 (520
mg, 1.020
mmol) in Et0H (9.8 mL) at room temperature. The reaction mixture was heated at
80 C for
27 h, after which it was cooled to room temperature. The reaction mixture was
transferred to
a separatory funnel, which was extracted with Et0Ac. The combined organic
extracts were
washed with water and brine, then dried over Na2SO4, filtered and
concentrated. The crude
product was purified by column chromatography (silica gel, 0% to 20% Et0Ac in
hexanes) to
give product 33 (387 mg, 72% yield) as a white solid: m/z 525.3 (M+1).
Compound 34: Na0Me (25 w/w% solution in Me0H, 0.12 mL, 0.525 mmol) was
added to a solution of compound 33 (128 mg, 0.244 mmol) in Me0H (1.2 mL) at
room
temperature. The reaction was then heated to 55 C and stirred for 1 h. After
cooling to room
temperature, the reaction was diluted with t-BuOMe (3 mL) and was cooled to 0
C. 1 N HC1
(aq) (5 mL) was added. After stirring for another 5 min, the reaction mixture
was transferred
to a separatory funnel, which was extracted with Et0Ac. The combined Et0Ac
extracts were
washed with water, dried over Na2SO4, filtered, and concentrated to give
product 34 (131 mg)
as a white solid: m/z 525.3 (M+1). Compound 34 is an isomeric mixture of C3
ketone and
enol forms.
Compound 63295: 1,3-Dibromo-5,5-dimethylhydantoin (40 mg, 0.140 mmol) in
DMF (0.5 mL) was added to a solution of compound 34 (126 mg, 0.240 mmol) in
DMF (1.6
mL) at 0 C. After stirring at 0 C for 40 min, the reaction was treated with
pyridine (40 1AL,
0.495 mmol), and was heated at 55 C for 7 h. After cooling to room
temperature, brine was
added, and the reaction mixture was transferred to a separatory funnel, which
was extracted
with Et0Ac. The combined organic extracts were washed with brine, 10%
Na2503(aq)
136
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
solution, 1 N HC1(aq), and water. The organic layer was separated, which was
dried over
Na2SO4, filtered, and concentrated. The crude product was purified by
column
chromatography (silica gel, 0% to 20% Et0Ac in hexanes) to give product 63295
(34 mg,
27% yield from 33) as a white solid: 1H NMR (400 MHz, CDC13) 6 7.83 (s, 1H),
3.68 (s, 3H),
3.36 (dd, 1H, J = 16.8, 4.8 Hz), 2.82-2.91 (m, 1H), 2.54 (d, 1H, J = 3.6 Hz),
1.76-2.06 (m,
4H), 1.52-1.74 (m, 6H), 1.04-1.50 (m, 8H), 1.20 (s, 3H), 1.15 (s, 3H), 1.13
(s, 3H), 0.93 (s,
3H), 0.92 (s, 6H), 0.90 (s, 3H); m/z 523.3 (M+1);
Compound 35: P0C13 (0.14 mL, 1.502 mmol) was added to a solution of compound
33 (200 mg, 0.381 mmol) in pyridine (1.9 mL) at room temperature. After
stirring for 5 h, the
reaction mixture was diluted with Et0Ac (5 mL), and was quenched with 1 N
HC1(aq) (5
mL). The reaction mixture was transferred to a separatory funnel, which was
extracted with
Et0Ac. The combined organic extracts were washed with 1 N HC1(aq) and brine,
then dried
over Na2SO4, filtered, and concentrated. The crude product was purified by
column
chromatography (silica gel, 0% to 50% Et0Ac in hexanes) to give product 35
(185 mg, 75%
yield) as a colorless glassy solid: m/z 525.4 (M+1);
Compound 36: Na0Me (25 w/w% solution in Me0H, 0.17 mL, 0.743 mmol) was
added to a solution of compound 35 (177 mg, 0.337 mmol) in Me0H (1.7 mL) at
room
temperature. The reaction was then heated to 55 C and stirred for 4 h. After
cooling to 0 C,
t-BuOMe and 1 N HC1(aq) were added, and the reaction mixture was stirred for 5
min. The
reaction mixture was then transferred to a separatory funnel, which was
extracted with
Et0Ac. The combined Et0Ac extracts were washed with 1 N HC1(aq) and brine,
then dried
over Na2SO4, filtered, and concentrated to give product 36 (164 mg, 93% yield)
as a white
solid: m/z 525.4 (M+1). Compound 36 is an isomeric mixture of C3 ketone and
enol forms.
Compound 63296: 1,3-Dibromo-5,5-dimethylhydantion (53 mg, 0.185 mmol) in
DMF (0.8 mL) was added to a solution of compound 36 (163 mg, 0.311 mmol) in
DMF (2.1
mL) at 0 C. After stirring at 0 C for 1 h, the reaction was treated with
pyridine (50 [iL,
0.618 mmol) and was heated at 55 C for 23 h. After cooling to room
temperature, brine was
added, and the reaction mixture was transferred to a separatory funnel, which
was extracted
with Et0Ac. The combined organic extracts were washed with brine, 10%
Na2503(aq)
solution, 1 N HC1(aq), and water, then dried over Na2504, filtered, and
concentrated to give
product 63296 (150 mg, 93% yield) as a white solid: 1H NMR (400 MHz, CDC13) 6
7.88 (s,
1H), 5.57 (d, 1H, J= 4.8 Hz), 4.06 (t, 1H, J= 6.0 Hz), 3.73 (s, 3H), 2.68 (dd,
1H, J = 14.4,
10.4 Hz), 2.48-2.60 (m, 1H), 2.13 (d, 1H, J = 14.4 Hz), 1.65-1.87 (m, 3H),
1.19-1.64 (m,
137
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
13H), 1.17 (s, 3H), 1.13 (s, 3H), 1.11 (s, 3H), 1.06 (s, 3H), 1.00 (s, 3H),
0.97 (s, 3H), 0.88 (s,
3H); m/z 523.3 (M+1).
Compound 37: m-CPBA (77%, 7.04 g, 31.52 mmol) was added to a solution of
compound 402-49 (1.60 g, 3.15 mmol) in CH2C12 (28 mL) at room temperature.
After
stirring for 8 h, additional m-CPBA (77%, 3.52 g, 15.71 mmol) was added, and
the reaction
was stirred for another 40 h. Na2S03 (aq.) solution was then added. After
another 10 min,
the reaction mixture was transferred to a separatory funnel, which was
extracted with Et0Ac.
The combined Et0Ac extracts were washed with NaHCO3 (aq.) solution, dried over
MgSO4,
filtered, and concentrated. The crude product was purified by column
chromatography (silica
gel, 0% to 45% Et0Ac in hexanes) to give product 37 (358 mg, 22% yield) as a
white foam
solid: m/z 526.3 (M+1).
Compound 38: Na0Me (25 w/w% solution in Me0H, 20 [iL, 0.087 mmol) was added
to a solution of compound 37 (38 mg, 0.072 mmol) in Me0H (0.7 mL) at room
temperature.
The reaction was then heated to 55 C, and stirred for 2 h. After cooling to 0
C, t-BuOMe
and 1 N (aq.) HC1 were added. The reaction mixture was then transferred to a
separatory
funnel, which was extracted with Et0Ac. The combined Et0Ac extracts were
washed with
water, dried over MgSO4, filtered, and concentrated. The crude product was
purified by
column chromatography (silica gel, 0% to 50% Et0Ac in hexanes) to give product
38 (25 mg,
66% yield) as a white foam solid: m/z 526.4 (M+1). Compound 38 is an isomeric
mixture of
C3 ketone and enol forms.
Compound 63263: A solution of 1,3-dibromo-5,5-dimethylhydantion (6.9 mg, 0.024
mmol) in DMF (0.2 mL) was added to a solution of compound 38 (25 mg, 0.048
mmol) in
DMF (0.8 mL) at 0 C. After stirring at 0 C for 1 h, the reaction was treated
with pyridine
(12 [it, 0.15 mmol), and was heated at 55 C for 3 h. After cooling to room
temperature, the
reaction was diluted with Et0Ac, and transferred to a separatory funnel, which
was then
washed with Na2503 (aq.) solution, 1N (aq.) HC1 and water. The organic extract
was
separated, dried over Mg504, filtered, and concentrated. The crude product was
purified by
column chromatography (silica gel, 0% to 45% Et0Ac in hexanes) to give product
63263 (18
mg) as a white foam solid: 1H NMR (400 MHz, CDC13) 6 7.83 (s, 1H), 4.95 (d,
1H, J = 7.2
Hz), 3.73 (s, 3H), 2.79 (m, 2H), 2.37 (d, 1H, J = 14.4 Hz), 1.92 (m, 2H), 1.77
(d, 1H, J = 10.4
Hz), 1.44-1.74 (m, 8H), 1.20-1.41 (m, 5H), 1.19 (s, 3H), 1.15 (s, 3H), 1.13
(s, 3H), 1.08 (s,
3H), 1.07 (s, 3H), 0.96 (s, 3H), 0.89 (s, 3H); m/z 524.3 (M+1).
Compound 39: LiA1H4 (2.0 M in THF, 48 1AL, 0.096 mmol) was added to a solution
of compound 37 (50 mg, 0.095 mmol) in THF (0.95 mL) at 0 C. After stirring
for 40 min,
138
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
the reaction was quenched by adding water (1 mL) carefully. After stirring at
room
temperature for 10 min, the reaction mixture was transferred to a separatory
funnel, which
was extracted with Et0Ac. The combined organic extracts were washed with 1N
(aq.) HC1,
and water, dried over MgSO4, filtered, and concentrated. The crude product was
purified by
column chromatography (silica gel, 0% to 40% Et0Ac in hexanes) to give product
39 (31 mg,
62% yield) as a white foam solid: m/z 528.3 (M+1). The stereochemical
configuration of
C12 was not assigned.
Compound 40: Na0Me (25 w/w% solution in Me0H, 16 [iL, 0.070 mmol) was added
to a solution of compound 39 (30 mg, 0.057 mmol) in Me0H (0.6 mL) at room
temperature.
The reaction was then heated to 55 C, and stirred for 2 h. After cooling to 0
C, t-BuOMe
and 1 N (aq.) HC1 were added. The reaction mixture was then transferred to a
separatory
funnel, which was extracted with Et0Ac. The combined Et0Ac extracts were
washed with
water, dried over MgSO4, filtered, and concentrated. The crude product was
purified by
column chromatography (silica gel, 0% to 40% Et0Ac in hexanes) to give product
40 (25 mg,
83% yield) as a white foam solid: m/z 510.3 (M-18+1). Compound 40 is an
isomeric mixture
of C3 ketone and enol forms. The stereochemical configuration of C12 was not
assigned.
Compound 63289: 1,3-Dibromo-5,5-dimethylhydantion (6.8 mg, 0.024 mmol) was
added to a solution of compound 40 (25 mg, 0.047 mmol) in DMF (0.47 mL) at 0
C. After
stirring at 0 C for 1 h, the reaction was treated with pyridine (12 [LL, 0.15
mmol), and was
heated at 55 C for 3 h. After cooling to room temperature, the reaction was
diluted with
Et0Ac, and transferred to a separatory funnel, which was then washed with
Na2S03 (aq.)
solution, 1N (aq.) HC1 and water. The organic extract was separated, dried
over Mg504,
filtered, and concentrated. The crude product was purified by column
chromatography (silica
gel, 0% to 40% Et0Ac in hexanes) to give partially purified product 63289 (16
mg), which
was purified again by preparative TLC plate (silica gel, eluted with 8% Et0Ac
in hexanes) to
give product 63289 (10 mg, 40% yield) as a white foam solid: 1H NMR (400 MHz,
CDC13) 6
7.64 (s, 1H), 5.12 (m, 1H), 4.33 (d, 1H, J= 6.8 Hz), 3.71 (s, 3H), 2.54 (m,
1H), 2.43 (d, 1H, J
= 2.8 Hz), 1.21-1.96 (m, 18H), 1.19 (s, 6H), 1.13 (s, 3H), 1.05 (s, 3H), 1.02
(s, 3H), 0.95 (s,
3H), 0.88 (s, 3H); m/z 508.3 (M-18+1). The stereochemical configuration of C12
was not
assigned.
Example 4 ¨Uptake of 404-02 into CNS and Lungs of Monkeys
Plasma concentrations following oral dosing: Compound 404-02 shows high uptake
in the CNS and lung in monkeys following oral dosing: 2 male and 2 female
cynomolgus
139
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
monkeys were administered 404-02 at 0.5, 5, 25 or 75 mg/kg/day doses via oral
gavage.
Doses were prepared in sesame oil and individualized to body weight on the day
of dosing.
Blood was drawn prior to dosing and at 1, 2, 4, 8 and 24 hours post-dose on
days 1 and 12.
Blood samples were collected from the femoral artery/vein for determination of
404-02
plasma concentrations. Blood was placed in tubes containing K3EDTA and stored
on ice until
centrifugation at room temperature. The isolated plasma was transferred to
cryovials and
stored at -80 C until sample processing and LC-MS/MS analysis. Extracted
plasma standard
curves were prepared from fresh stock solutions and analyzed prior to study
samples.
Summary results are shown in Tables 2a and 2b.
Population mean pharmacokinetic parameter estimates were obtained by
performing
non-compartmental analysis of the 404-02 plasma concentration-versus-time data
using
WinNonlinTM software version 5.2. Across the investigated dose range, 404-02
demonstrated
dose-dependent kinetics with increased oral clearance (Cl/F), reciprocal
shortening of
elimination half-life (T112) and an increasing apparent volume of distribution
(Vz/F) with
increasing dose. A 1.6-fold increase in the area under the concentration
versus time curve
over 24 hours (AUCo-240 was observed at the 75 mg/kg dose level after 12 days
of dosing
compared to the corresponding AUC on day 1. Accumulation was not observed at
any of the
other dose levels. The observed mean maximum plasma concentration (C.) for the
0.5, 5,
and 75 mg/kg/day dose groups on day 12 were 4.6, 12.7, 17.5 and 48.6 nM 404-
02,
20 respectively.
Table 2a shows the population mean plasma pharmacokinetics of 404-02 in
cynomolgus monkey on day 1 of study (n=4). Pharmacokinetic parameters obtained
using
non-compartmental analysis, WinNonlinTM version 5.2.
Table 2a: Day 1 Plasma Pharmacokinetics of 404-02:
Mean Maximum 404-02
Dose Mean Maximum 404-02 Plasma
Plasma Concentration (nM)
(mg/kg/d) Concentration (ng/mL) SEM
SEM
0.5 1.95 0.35 3.4 0.61
5 10.1 3.8 17.6 6.6
25 16.8 1.3 29.3 2.3
75 19.4 2.7 33.8 4.7
140
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
Table 2b shows the population mean plasma pharmacokinetics of 404-02 in
cynomolgus monkey on day 12 of study (n=4). Pharmacokinetic parameters were
obtained
using non-compartmental analysis using WinNonlinTM version 5.2 software.
Table 2b: Day 12 Plasma Pharmacokinetics of 404-02:
Mean Maximum 404-02
Dose Mean Maximum 404-02 Plasma
Plasma Concentration (nM)
(mg/kg/d) Concentration (ng/mL) SEM
SEM
0.5 2.65 0.6 4.6 1.0
7.3 1.7 12.7 2.9
25 10.0 3.7 17.5 6.4
75 28.0 12.2 48.6 12.2
5
CNS and lung concentrations following oral dosing: 2 male and 2 female
cynomolgus monkeys were administered 404-02 at 0.5, 5, 25 or 75 mg/kg/day
doses via oral
gavage, in addition to a control (non-treatment) group with 2 animals per sex.
Animals were
sacrificed approximately 3 hours after dosing on day 15 following harvest of
brain and lung
tissues. Each sample collected was rinsed in 1 x isotonic phosphate buffered
saline and
blotted dry before weighing. Harvested tissue slices were transferred to
cryovials and stored
at -80 C until processing and LC-MS/MS analysis. Standard curves were derived
for 404-02
in homogenates of these tissues and were used to quantify the day 15 samples.
The concentration of 404-02 necessary for 50% suppression of nitric oxide (NO)
production in macrophages stimulated with interferon-gamma is approximately 45
nM. As
evidenced by the data, 0.5 mg/kg administered orally as the lowest dose in
this study for 15
days resulted in a mean 404-02 CNS concentration of 2,162 nM, which markedly
exceeds the
IC50 value for NO production in vitro. The CNS penetration provides a large
therapeutic
margin at all doses tested (Table 2c). For instance, the mean 404-02 CNS
concentrations
following administration of 0.5, 5, 25 and 75 mg/kg/day 404-02 indicates a 48-
, 44-, 37- and
75-fold excess compared to the dose necessary to suppress inflammation in
vitro. For
comparison, the mean 404-02 lung tissue exposure at 75 mg/kg/day 404-02 showed
a 133-
fold increase (Table 2d). Non-linear disposition of 404-02 in CNS and lung
tissue was
observed suggesting 404-02 is taken up by a saturable mechanism(s) for
transport across
membranes and/or intracellular binding. In addition, concentrations of 404-02
in monkey
CNS and lung tissue exceed plasma levels. Table 2c shows the population mean
404-02
141
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
cynomolgus monkey CNS tissue exposure on day 15. Table 2d shows the population
mean
404-02 cynomolgus monkey lung tissue content on day 15.
Table 2c: Day 15 CNS Tissue Content/Concentration of 404-02:
D Mean 404-02 CNS Mean
404-02 CNS
ose
Tissue Content Tissue Concentration
(mg/kg/day)
(ng/g) SEM (nM)* SEM
0.5 1198 712 2087
1239
1090 564 1900 982
25 930 352 1618
612
75 1898 496 3305
864
* Conversion based on the assumption the density of tissue is equal to water,
1 g/mL.
5 Table 2d: Day 15 Lung Tissue Content/Concentration of 404-02:
Mean 404-02 Lung Mean
404-02 Lung
Dose Level
Tissue Content
Tissue Concentration
(mg/kg/day)
(ng/g) SEM (nM)*
SEM
0.5 705 292 1227 508
5 24 15 55 27
25 141 50 246 87
75 3405 824 5929 1435
Example 5 ¨Uptake of 404-02 into CNS and Lungs of Rats
Compound 404-02 reaches high concentrations in the lung and CNS of rats
following
oral dosing: To assess basic pharmacokinetic parameters following oral dosing,
9 male and 9
female Sprague Dawley (SD) rats were administered 404-02 at 1, 10 or 50 mg/kg
doses via
oral gavage. Doses were prepared in sesame oil and individualized to body
weight on the day
of dosing. Blood was drawn at 0, 1, 2, 4, 8 and 24 hours post-dose on days 1
and 15. Blood
was collected from the orbital sinus after carbon dioxide/oxygen inhalation
for determination
of 404-02 plasma concentrations. Plasma was transferred to cryovials and
stored at -80 C
until processing and LC-MS/MS analysis. Summary results for Day 1 and Day 15
are shown
in Tables 2a and 2b, respectively. A standard curve was derived for 404-02 in
rat plasma, and
quantification of experimental results was based on this standard curve.
142
CA 02721837 2010-10-15
WO 2009/129545 PCT/US2009/041170
Table 3a shows population mean plasma pharmacokinetics of 404-02 in SD rats on
day 1 of study (n=9/sex/dose level). Pharmacokinetic parameters were obtained
using non-
compartmental analysis, WinNonlinTM version 5.2.
Table 3a: Day 1 Pharmacokinetic Parameters
Mean Maximum 404-02
Dose Mean Maximum 404-02 Plasma
(mg/kg/day) Concentration (ng/mL) SEM Plasma
Concentration (nM)
SEM
1 3.3 0.65 5.7 1.1
66 16.5 116 28.7
50 713 98.2 1241 171
5
Table 3b shows the population mean plasma pharmacokinetics of 404-02 in SD
rats
on day 15 of study (n=9/sex/dose level). Pharmacokinetic parameters were
obtained using
non-compartmental analysis, WinNonlinTM version 5.2.
Table 3b: Day 15 Pharmacokinetic Parameters:
Mean Maximum 404-02
Dose Mean Maximum 404-02 Plasma
(mg/kg/day) Concentration (ng/mL) SEM Plasma
Concentration (nM)
SEM
1 6.3 0.9 11.0 1.6
10 144 24.3 210 42.3
50 1129 114 1966 199
To examine tissue concentrations following oral dosing, 5 male and 5 female
Sprague
10
Dawley (SD) rats were administered 404-02 at 1, 10, 50 or 150 mg/kg/day via
oral gavage.
Doses were prepared in sesame oil and individualized to body weight on the day
of dosing.
Animals were sacrificed 3 hours post-dose on day 15 of study following harvest
of brain and
lung specimens. Each sample collected was rinsed in lx isotonic phosphate
buffered saline
and blotted dry before weighing. Tissue slices were transferred to cryovials
and stored at -
80 C until processing and LC-MS/MS analysis. Summary results for CNS and lung
samples
are shown in Tables 3c and 3d, respectively. Standard curves were derived for
404-02 in
these tissues.
143
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
Table 3c: Population Mean 404-02 CNS Tissue Content in SD Rats on Day 15.
Dose Mean 404-02 CNS Tissue Mean 404-02 CNS Tissue
(mg/kg/day) Content (ng/g = ng/mL)* SEM
Concentration (nM) SEM
1 29 6.8 51 11.8
421 99 733 173
50 750 108 1306 188
150 640 82 1114 143
* Conversion based on the assumption the density of tissue is equal to water,
1 g/mL.
Table 3d: Population Mean 404-02 Lung Tissue Content in SD Rats on Day 15.
Dose Mean dh404 Lung Tissue Content Mean dh404 Lung
Tissue
(mg/kg/day) (ng/g = ng/mL)* SEM
Concentration (nM) SEM
1 558 123 972 215
10 4032 928 7020 1616
50 7165 1221 12474 2126
150 9719 1643 16921 2860
* Conversion based on the assumption the density of tissue is equal to water,
1 g/mL.
5 Example 6 ¨ Rodent Toxicity Comparison between 402 and 402-02
A study was performed in Sprague Dawley rats using both 402 and 402-02.
Animals
were dosed orally once daily for 7 days. The low dose 402 group had elevated
total bilirubin
and GGT levels as well as suppressed weight gain. The high dose animals that
were treated
with 402 were all sacrificed in extremis on Day 6 before study completion. GGT
and total
10 bilirubin levels were elevated in these animals as well. However, no
toxicity as assessed by
clinical observations, weight gain, GGT, and total bilirubin was observed in
any animal
treated with 402-02 (Table 4). In a second study involving oral administration
to Sprague-
Dawley rats for 14 days, 402-02, achieved comparable blood levels to that of
402. However,
no significant toxicity was observed as assessed by weight loss, clinical
observations, and
GGT and total bilirubin elevations relative to controls at doses up to 1,500
mg/m2/day for 14
days, which is 50-fold higher than the MTD of RTA 402 in this species (Table
2).
144
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
Table 4: Comparing Compounds 402-02 and 402 for Rodent Toxicity
Weight GGT Total
Timepoint/ Dose Level Survival vs U/L
( ) Bilirubin
Control (mg/dL)
7 Day Study ¨ Crystalline Forms of 402 and 402-02
Vehicle Control 4/4(100%) 100% < 5 0.13
402 - 60 mg/m2/day 4/4 (100%) 35% 8.7 0.25
402 - 180 mg/m2/day 0/4 (0%) NA 5.0 0.78
402-02 - 60 mg/m2/day 4/4 (100%) 106% < 5 0.20
402-02 - 180 mg/m2/day 4/4 (100%) 132% < 5 0.20
14 Day Study ¨ Crystalline Form of 402-02
Vehicle Control 10/10 (100%) 100% < 5 0.20
402-02 - 60 mg/m2/day 10/10 (100%) 95% < 5 0.24
402-02 - 180 mg/m2/day 5/5 (100%) 102% < 5 0.22
402-02 - 600 mg/m2/day 5/5 (100%) 96% < 5 0.20
402-02 - 1,500 mg/m2/day 5/5 (100%) 105% < 5 0.20
Example 7 ¨Toxicity Comparison in Mice
In this study, six compounds (401, 402, 404, 401-2, 402-2, and 404-2) were
assessed
for toxicity in mice in a 14-day study. Each compound was formulated in sesame
oil and
administered daily by oral gavage at doses of 10, 50, 100, or 250 mg/kg (n = 4
per group). At
higher doses (above 10 mg/kg/day) both 401 and 402 caused at least 50%
mortality; 404 was
non-toxic. In contrast, no mortality was observed in the 402-2 and 404-2
groups and only the
highest dose of 401-02 caused any lethality (Table 5). Body weight
measurements (FIGS.
29-31) were consistent with the mortality observations. The two highest doses
of 401 and
402 were lethal within 4 days, in contrast to the effects of 401-2 and 402-2.
145
CA 02721837 2010-10-15
WO 2009/129545 PCT/US2009/041170
Table 5: Mortality Observations in 14-Day Toxicity Study
Dose Number
Group Compound Schedule N Comments
(mg/kg) of Deaths
1 vehicle QDx14, D1-
4 0
14
2 401 10 QDx14, D1- 4 0
14
3 401 50 QDx14, D1-
4 2
14
4 401 100 QDx14, D1-
4 4
14
401 250 QDx14, D1-
4 4
14
6 401-02 10 QDx14, D1- 4 0
14
7 401-02 50 QDx14, D1- 4
14 1* *Due
to gavage injury
8 401-02 100 QDx14, D1- 4 0
14
9 401-02 250 QDx14, D1- 4
1 Sacrificed due to
14
weightless on Day 11
402 10 QDx14, D1- 4 0
14
11 402 50 QDx14, D1- 4
4
14
12 402 100 QDx14, D1- 4
4
14
13 402 250 QDx14, D1- 4
4
14
14 402-02 10 QDx14, D1- 4 0
14
402-02 50 QDx14,
4 0
ID1-14
15 402-02 100 QDx14, D1- 4 0
14
17 402-02 250 QDx14, D1- 4 0
14
la 404 10 QDx14, D1- 4 0
14
19 404 50 QDx14, D1- 4 0
14
404 100 QDx14, D1- 4 0
14
21 404 250 QDx14, D1- 4 0
14
22 404-02 10 QDx14, D1- 4 0
14
146
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
Dose Number
Group Compound Schedule N Comments
(mg/kg) of Deaths
D1-
23 404-02 50 QDx14, 4 0
14
D1-
22 404-02 100 QDx14, 4 0
14
D1-
23 404-02 250 QDx14, 4 0
14
In a second experiment, six additional compound differing only in the
saturation or
non-saturation of the C ring were tested for toxicity in mice by daily oral
administration for 9
days, using sesame oil as the vehicle. In this study, no significant toxicity
was observed. The
deaths of two animals were attributed to gavage errors during the
administration of the test
article. No significant differences in weight were observed in any group
compared to the
vehicle-treated controls. Results are summarized in Table 6 below. As with
compounds 402-
2, 401-2 and 404-2 above, compounds with saturation in the C ring consistently
show low
toxicity in rodents. Compounds lacking saturation in the C ring show
significant rodent
toxicity in some cases (e.g., 401 and 402). Predictably low rodent toxicity
provides an
advantage since high rodent toxicity can be a significant complication in
conducting
preclinical studies required for development and registration of therapeutic
compounds for
use in humans or non-human animals.
Table 6: Further Mouse Toxicity Results.
Compound Dose (per day, p.o.) Mortality
3 mg/kg 0/5
63112 10 mg/kg 0/5
30 mg/kg 1/5
3 mg/kg 0/5
63323 10 mg/kg 0/5
30 mg/kg 0/5
3 mg/kg 0/5
63324 10 mg/kg 0/5
30 mg/kg 0/5
147
CA 02721837 2010-10-15
WO 2009/129545 PCT/US2009/041170
Compound Dose (per day, p.o.) Mortality
3 mg/kg 0/5
63325 10 mg/kg 0/5
30 mg/kg 0/5
3 mg/kg 0/5
63166 10 mg/kg 0/5
30 mg/kg 0/5
3 mg/kg 0/5
63326 10 mg/kg 1/5
30 mg/kg 0/5
Example 8 ¨ Aqueous Solubility of Oleanolic Acid Derivatives
The aqueous solubility of the compounds shown here was determined using the
procedures outlined in Example 1.
Aqueous Solubility
Compound ID(s) Structure
(04)
0
63097 0
Air 0.CH3
(402) NC Aagri 0 1.46
i
0
63102 O
H
0 N CF 3
0.06
(dh404) NC i)i
0
0 riP
148
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
Aqueous Solubility
Compound ID(s) Structure
(11M)
.1
0 O
63198
A app NH2 163.6
NC
,
0 O
e=
63202 OH 1.89
NC Aggi i
0 7,7
,
0 O
63208 NH2 9.49
NC Adi3O
0 77
i
0 O
63214 00 NH2
112.2
NC,,,
0 7
o
OH O di 0,
63219 CH3 13.58
NC, A
0
0 7-
149
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
Aqueous Solubility
Compound ID(s) Structure
(PM)
0
63221 NC . OH 8.78
0010
OH
0
0
63226 Ny NC CF3
0.71
.0,410
0
0
0
63231 NC OH 1.23
0,0
0 ,
0
N
63232 0.75
NC so
0
CH3
= \ 0
63237 00 5.16
NC,,
0 7-
* * * * * * * * * * * * * * * *
150
CA 02721837 2015-07-30
All of the methods disclosed and claimed herein can be made and executed
without undue experimentation in light of the present disclosure. It will be
apparent that
certain agents which are both chemically and physiologically related may be
substituted
for the agents described herein while the same or similar results would be
achieved. The
scope of the claims should not be limited by the preferred embodiments and
examples,
but should be given the broadest interpretation consistent with the
description as a whole.
151
CA 02721837 2015-07-30
REFERENCES
The following references provide exemplary procedural or other details
supplementary to those set forth herein:
U.S. Patent 5,443,826
U.S. Patent 5,599,795
U.S. Patent 6,025,395
U.S. Patent 6,974,801
U.S. Patent Application Publication No. 2010-0048892
U.S. Patent Application Publication No. 2010-0048911
U.S. Patent Application Publication No. 2010-0283450
U.S. Patent Application Publication No.2010-0041904
U.S. Patent Application Publication No. 2009-0060873
U.S. Patent Application Publication No. 2009-0326063
U.S. Patent Application Publication No. 2009-0060873
U.S. Patent Application Publication No. 2012-0283450 by Eric Anderson, Gary L.
Bolton, Deborah Ferguson, Xin Jiang, Robert M. Kral, Jr., Patrick M.
O'Brian and Melean Visnick, entitled "Natural Products Including an Anti-
Inflammatory Pharmacore and Methods of Use," filed April 20, 2009.
U.S. Patent Application Publication No. 2010-0048892 by Eric Anderson, Xin
Jiang
and Melean Visnick, entitled "Antioxidant Inflammation Modulators:
Oleanolic Acid Derivatives with Amino and Other Modifications At C-17,"
filed April 20, 2009.
U.S. Patent Application Publication No. 2010-0048911 by Xin Jiang, Jack
Greiner,
Lester L. Maravetz, Stephen S. Szucs, Melean Visnick, entitled "Antioxidant
Inflammation Modulators: Novel Derivatives of Oleanolic Acid," filed April
20, 2009.
U.S. Patent Application Publication No. 2010-0041904 by Xin Jiang, Xioafeng
Liu,
Jack Greiner, Stephen S. Szucs, Melean Visnick entitled, "Antioxidant
Inflammation Modulators: C-17 Homologated Oleanolic Acid Derivatives,"
filed April 20, 2009.
152
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
Abraham and Kappas, Free Radic. Biol. Med., 39(1):1-25, 2005.
Adult Treatment Panel III, or ATP III
Ahmad et al., Cancer Res., 68(8):2920-2926, 2008.
Ahmad et al., J Biol Chem., 281(47):35764-35769, 2006.
Akiyama et al., Alzheimer Dis. Assoc. Disord., 14(1):S47-53, 2000.
Angulo et al., Eur. J. Immunol., 30:1263-1271, 2000.
Araujo et al., J. Immunol., 171(3):1572-1580, 2003.
Arend and Dayer, Arthritis Rheum., 38:151-160, 1995.
Arend et al., Annu. Rev. Immunol., 16:27-55, 1998.
Autenrieth et al., Infect. Immun., 62:2590-2599, 1994.
Bach, Hum. Immunol., 67(6):430-432, 2006
Bagasra et al., Proc. Natl. Acad. Sci. USA, 92:12041-12045, 1995.
Ball, Ann. Rheum. Dis., 30:213-223, 1971.
Beal, Curr. Opin. Neurobiol., 6:661-666, 1996.
Bendzen et al., Scand. J. Rheumatol., 28:599-606, 1988.
Blumberg et al., Arthritis Rheum., 7:93-97, 1964.
Botoman et al., Am. Fam. Physician, 57(1):57-68, 1998.
Brandt et al., Arthritis Rheum., 43:1346-1352, 2000.
Braun et al., Arthritis Rheum., 42:2039-2044, 1999.
Brewerton et al., Lancet., 1:904-907, 1973a.
Brewerton et al., Lancet., 1:956-957, 1973b.
Bronte et al., Trends Immunol., 24:302-306, 2003.
Brown and DuBois, J. Clin. Oncol., 23:2840-2855, 2005.
Brynskov et al., N. Engl. J. Med., 321(13):845-850, 1989.
Burger and Dayer, Neurology, 45(6S-6):S39-43, 1995.
Cai et al., Nat. Med., 11(2):183-190, 2005.
Calin and Taurog, In: The Spondylarthritides, Calin et al. (Eds.), Oxford, UK.
Oxford
University Press, 179, 1998.
Cann et al., Gut., 24(12):1135-1140, 1983.
Chauhan and Chauhan, Pathophysiology, 13(3):171-181 2006.
Chomarat et al., Arthritis Rheum., 38:1046-1054, 1995.
Coyle and Puttfarcken, Science, 262:689-695, 1993.
Crowell et al., Mol. Cancer Ther., 2:815-823, 2003.
Culver et al., Science, 256:1550-1552, 1992.
153
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
de Waal et al., J. Exp. Med., 174:1209-1220, 1991.
Dickerson et al., Prog Neuropsychopharmacol Biol. Psychiatry, March 6, 2007.
Dinarello, Int. Rev. Immunol., 16:457-499, 1998.
Dinkova-Kostova et al., Proc Natl Acad Sci USA, 102(12):4584-4589, 2005.
Dionne et al., Clin. Exp. Imunol., 112(3):435-442, 1998.
Doran et al., J. Rheumatol., 30(2):316-320, 2003.
Drossman et al., Dig. Dis. Sci., 38(9):1569-1580, 1993.
Drossman et al., Gastroenterol., 112(6):2120-2137, 1997.
Dudhgaonkar et al., Eur. J. Pain, 10(7):573-9, 2006.
Eikelenboom et al., Glia, 40(2):232-239, 2002.
Ettehadi et al., Clin. Exp. Immunol., 96(1):146-151, 1994.
Everhart et al., Gastroenterol., 100(4):998-1005, 1991.
Fearon and Locksley, Science, 272(5258):50-53, 1996.
Feldtkeller et al., Rheumatol. Int., 23(2):61-66, 2003.
Firestein et al., Arthritis Rheum., 37:644-652, 1994.
Forstermann, Biol. Chem., 387:1521, 2006.
Fujikawa et al., Ann. Rheum. Dis., 54:318-320, 1995.
Funakoshi et al., Digestion, 59(1):73-78, 1998.
Galley and Webster, Br. J. Anaesth.,77 :11-16, 1996.
Gehrmann et al., Glia, 15(2):141-151, 1995.
Genain and Nauser, J. MoL Med., 75:187-197, 1997.
Gladman et al., Br. J. Rheumatol., 22:675-679, 1995.
Gladman et al., J. Med., 62:127-141, 1987.
Gladman, Rheum. Dis. Clin. North Am., 18:247-256, 1992.
Goodman et al., Kidney Int., 72(8):945-953, 2007.
Graeber et al., Glia, 40(2):252-259, 2002.
Greten et al., Cell, 118:285-296, 2004.
Griffin et al., Proc. Natl. Acad. Sci. USA, 86(19):7611-7615, 1989.
Guilherme et al., Nat. Rev. Mol. Cell Biol., 9(5):367-77, 2008.
Gwee et al., Gut., 44(3):400-406., 1999.
Hahn and Tsao, In: Dubois' Lupus Erythematosus, 4th Ed, Wallace and Hahn
(Eds.), Lea and
Febiger, Philadelphia, 195-201, 1993.
Handbook of Pharmaceutical Salts: Properties, and Use (Stahl & Wermuth eds.,
Verlag
Helvetica Chimica Acta, 2002.
154
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
Handbook of Pharmaceutical Salts: Properties, Selection and Use (Stahl &
Wermuth, Eds.),
Verlag Helvetica Chimica Acta, 2002.
Hannum et al., Nature, 343:336-340, 1990.
Hanson et al., BMC Medical Genetics, 6(7), 2005.
Hansson et al., Annu. Rev. Pathol. Mech. Dis., 1:297-329, 2006.
Harrison and Symmons et al., Ann. Rheum. Dis., 57(6):375-377, 1998.
Harrison et al., J. Rheumatol., 25(12):2324-2330, 1998.
Hart et al., Immunology, 84:536-542, 1995.
Hohler et al., Arthritis Rheum., 41:1489-1492, 1998.
Hohler et al., J. Invest. Dermatol., 109:562-565, 1997.
Honda et al., Bioorg. Med. Chem. Lett., 12:1027-1030, 2002.
Honda et al., Bioorg. Med. Chem. Lett., 19:2711-2714, 1998.
Honda et al., Bioorg. Med. Chem. Lett., 9:3429-3434, 1999.
Honda et al., J. Med. Chem., 43:1866-1877, 2000a.
Honda et al., J. Med. Chem., 43:4233-4246, 2000b.
Horwitz and Fisher, N. Engl. J. Med., 344(24):1846-1850, 2001.
Hotamisligil, Nature, 444(7121):860-7, 2006.
Ishikawa et al., Circulation, 104(15):1831-1836, 2001.
Ishizawa and Dickson, J. Neuropathol. Exp. Neurol., 60(6):647-657, 2001.
Jacob et al., Proc. Natl. Acad. Sci. USA, 87:1233-1237, 1990.
Jailwala et al., Ann. Intern. Med., 133(2):136-147, 2000.
Jarvis, Curr. Opin. Rheumatol., 10(5):459-467, 1998.
Jarvis, Pediatr. Ann., 31(7):437-446, 2002.
Jones et al., Br. J. Rheumatol., 33(9):834-839, 1994.
Jonsson et al., Br. J. Rheumatol., 32(7):578-581 1993.
Jonsson et al., Oral Dis., 8(3):130-140, 2002.
Jonsson et al., Trends Immunol., 22(12):653-654 , 2001.
Kahle et al., Ann. Rheum. Dis., 51:731-734, 1992.
Kaltschmidt et al., Proc. Natl. Acad. Sci. USA, 94:2642-2647, 1997.
Kawakami et al., Brain Dev., 28(4):243-246, 2006.
Kellow and Phillips, Gastroenterol., 92(6):1885-1893, 1987.
Kendall-Tackett, Trauma Violence Abuse, 8(2):117-126, 2007.
Khan et al., J. Neurochem., 71:78-87, 1998.
Khan et al., Toxicol. Applied Pharmacol., 103:482-490, 1990.
155
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
Kortylewski et al., Nat. Med., 11:1314-1321, 2005.
Kotake et al., Infect. Immun., 67:2682-2686, 1999.
Kotzin and O'Dell, In: Samler's Immunologic Diseases, 5th Ed., Frank et al.
(Eds.), Little
Brown & Co., Boston, 667-697, 1995.
Kotzin, Cell, 85:303-306, 1996.
Kruger et al., J. Pharmacol. Exp. Ther., 319(3):1144-1152, 2006.
Kuboyama, Kurume Med. J., 45(1):33-37, 1998.
Lahesmaa et al., J. Immunol., 148:3079-3085, 1992.
Lee et al., Glia., 55(7):712-22, 2007.
Lencz et al., Mol. Psychiatry, 12(6):572-80, 2007.
Liby et al., Nat Rev. Cancer, 7(5):357-369, 2007.
Lipsky, In: Harrison's principles of internal medicine, Fauci et a/.(Eds.),
14th Ed., NY,
McGraw-Hill, 1880-1888, 1998.
Liu et al., FASEB J., 20(2):207-216, 2006.
Lo et al., Curr. Dir. Autoimmun., 1:226-246, 1999.
Lugering et al., Ital. J. Gastroenterol. Hepatol., 30(3):338-344, 1998.
Lynn and Friedman, N. Engl. J. Med., 329(26):1940-1945, 1993.
Macatonia et al., J. Immunol., 150:3755-3765, 1993.
March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure,
2007.
March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure
(March's
Advanced Organic Chemistry), Smith and March (Eds.), 2007.
Marsal et al., Rheumatology, 38:332-337, 1999.
Mazur et al., Cell Microbiol., 9(7):1683-94, 2007.
Mazzoni et al., J. Immunol., 168:689-695, 2002.
McAlindon et al., Gut, 42(2):214-219, 1998.
McGeer and McGeer, Brain Res. Brain Res. Rev., 21:195-218, 1995.
McGeer et al., Neurology, 19:331-338, 1996.
McGonagle et al., Arthritis Rheum., 41:694-700, 1998.
McGonagle et al., Curr. Opin. Rheumatol., 11:244-250, 1999.
McIver et al., Pain, 120(1-2):161-9, 2005.
Mease et al., Lancet, 356:385-390, 2000.
Merrill and Benvenist, Trends Neurosci., 19:331-338, 1996.
Mertz et al., Gastroenterol., 118(5):842-848, 2000.
Moll and Wright, Ann. Rheum. Dis., 32:181-201, 1973.
156
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
Moll and Wright, Semin. Arthritis Rheum., 3:55-78, 1973.
Morris et al., J. Mol. Med., 80(2):96-104, 2002.
Morse and Choi, Am. J. Respir. Crit. Care Med., 172(6):660-670, 2005.
Morse and Choi, Am. J. Respir. Crit. Care Med., 27(1):8-16, 2002.
Nath et al., Neurology, 66(1):149-150, 2006.
National Institutes of Health, NIH Publication No. 01-3670, 2001.
Neal et al., BMJ., 314(7083):779-782, 1997.
Nichols, Drug News Perspect., 17(2):99-104, 2004.
Nielen et al., Arthritis Rheum., 50(2):380-386, 2004.
Ohnishi et al., Int. Immunol., 6:817-830, 1994.
Pall, Med. Hypoth., 69:821-825, 2007.
Partsch et al., Br. J. Rheumatol., 24:518-523, 1997.
Pica et al., Antimicrob Agents Chemother., 44(1):200-4, 2000.
Pimentel et al., Am. J. Gastroenterol., 95(12):3503-3506, 2000.
Pociot et al., Scand. J. Immunol., 42(4):501-504, 1995.
Prieur et al., Lancet., 2:1240-1242, 1987.
Rajakariar et al., Proc. Natl. Acad. Sci. USA, 104(52):20979-84, 2007.
Rantapaa-Dahlqvist et al., Arthritis Rheum., 48(10):2741-2749, 2003.
Reimund et al., Eur. J. Clin. Invest., 28(2):145-150, 1998.
Ribbens et al., Eur. Cytokine Netw.,11:669-676, 2000.
Rogers et al., Neurobiol Aging, 9(4):339-349, 1988.
Rogler and Andus, World J. Surg., 22(4):382-389, 1998.
Rooney et al., Rheumatol. Int., 10:217-219, 1990.
Ross et al., Nutr. Neurosci., 6(5):277-81, 2003.
Rostom et al., Ann. Intern. Med., 146, 376-389, 2007.
Rothstein, Med. Clin. North Am., 84(5):1247-1257, 2000.
Ruster et al., Scand. J. Rheumatol., 34(6):460-3, 2005.
Sacerdoti et al., Curr Neurovasc Res. 2(2):103-111, 2005.
Saiki et al., Scand. J. Gastroenterol., 33(6):616-622, 1998.
Salomonsson and Jonsson, Arthritis Rheum., 48(11):3187-3201, 2003.
Salomonsson et al., Scand. J. Immunol., 55(4):336-342, 2002.
Salvarani et al., Curr. Opin. Rheumatol. 1998; 10:299-305, 1998.
Salvemini et al., J. Clin. Invest., 93:1940-1947, 1994.
Sandler, Gastroenterol., 99(2):409-415, 1990.
157
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
Sarchielli et al., Cephalalgia, 26(9):1071-1079 , 2006.
Satoh et al., Proc. Natl. Acad. Sci. USA, 103(3):768-773, 2006.
Schellekens et al., Arthritis Rheum., 43(1):155-163, 2000.
Schlaak et al., Clin. Exp. Rheumatol., 14:155-162, 1996.
Schlaak et al., Eur. J. Immunol., 22:2771-2776, 1992.
Schlosstein et al., NE J. Medicine, 288:704-706, 1973.
Schreiber, Neth. J. Med., 53(6):524-31, 1998.
Schulz et al., Antioxid. Redox. Sig., 10:115, 2008.
Sieper and Braun, Arthritis Rheum., 38:1547-1554, 1995.
Simon et al., Clin. Exp. Immunol., 94:122-126, 1993.
Simon et al., Proc. Natl. Acad. Sci. USA, 91:8562-85666, 1994.
Simonian and Coyle, Annu. Rev. Pharmacol. Toxicol., 36:83-106, 1996.
Sinha et al., Cancer Res., 67:4507-4513, 2007.
Stack et al., Lancet, 349(9051):521-524, 1997.
Stewart et al., Neurology, 48:626-632, 1997.
Strejan et al., J. Neuroimmunol., 7:27, 1984.
Szabo et al., Nature Rev. Drug Disc., 6:662-680, 2007.
Takahashi et al., Cancer Res., 57:1233-1237, 1997.
Talley et al., Gastroenterol., 109(6):1736-1741, 1995.
Tamir and Tannenbaum, Biochim. Biophys. Acta., 1288:F31¨F36, 1996.
Targan et al., N. Engl. J. Med., 337(15):1029-1035, 1997.
Third Report of the National Cholesterol Education Program Expert Panel on
Detection,
Evaluation and Treatment of High Blood Cholesterol in Adults (Adult Treatment
Panel III, or ATP III), National Institutes of Health, 2001, NIH Publication
No. 01-
3670.
Touzani et al., J. Neuroimmunol., 100(1-2):203-215, 1999.
Tumlin et al., Am. J. Cardiol., 98(6A):14K-20K, 2006.
van den Berg, Semin. Arthritis Rheum., 30(5S-2):7-16, 2001.
van Dullemen et al., Gastroenterol., 109(1):129-135, 1995.
van Hogezand and Verspaget, Drugs, 56(3):299-305, 1998.
Vazquez et al., J. Virol., 79(7):4479-91, 2005.
Vodovotz et al., In; Handbook of Experimental Immunology, Volumes I¨IV, 1996.
Wardle, Nephrol. Dial. Transplant., 16(9):1764-8, 2001.
Warrington et al., Arthritis and Rheumatism, 44:13-20, 2001.
158
CA 02721837 2010-10-15
WO 2009/129545
PCT/US2009/041170
Weyand and Goronzy, Ann. NY Acad. Sci., 987:140-149, 2003.
Whitehead et al., Gastroenterol., 98(5 Pt 1):1187-1192, 1990.
Williams et al., Clin. Neurosci., 2(3-4):229-245, 1994.
Wordsworth, In: Genes and Arthritis, Brit. Medical Bulletin, 51:249-266, 1995.
Wright, Ann. Rheum. Dis., 15:348-356, 1956.
Wright, Clin. Orthop. Related Res., 143:8-14, 1979.
Xanthou et al., Arthritis Rheum., 44(2):408-418, 2001.
Yates et al., Cancer Res., 66(4): 2488-2494, 2006.
Yin et al., Arthritis Rheum., 40:1788-1797, 1997.
Yin et al., Rheumatology, 38:1058-1067, 1999.
Yoh et al., Kidney Int., 60(4):1343-1353, 2001.
Yu et al., Nat. Rev. Immunol., 7:41-51, 2007.
Zhou et al., Am. J. Pathol., 166(1):27-37, 2005.
Zhou et al., Cancer Sci., 98:882-889, 2007.
Zingarelli et al., J. Immunol., 171(12):6827-6837, 2003.
159
