U.S. patent application number 12/228750 was filed with the patent office on 2009-04-16 for heterocyclic inhibitors of necroptosis.
Invention is credited to Gregory D. Cuny, Alexei Degterev, John A. Porco, JR., Xin Teng, Junying Yuan.
Application Number | 20090099242 12/228750 |
Document ID | / |
Family ID | 40351022 |
Filed Date | 2009-04-16 |
United States Patent
Application |
20090099242 |
Kind Code |
A1 |
Cuny; Gregory D. ; et
al. |
April 16, 2009 |
Heterocyclic inhibitors of necroptosis
Abstract
The invention features a series of heterocyclic derivatives that
inhibit tumor necrosis factor alpha (TNF-.alpha.) induced
necroptosis. The heterocyclic compounds of the invention are
described by Formulas (I) and (Ia)-(Ie) and are shown to inhibit
TNF-.alpha. induced necroptosis in FADD-deficient variant of human
Jurkat T cells. The invention further features pharmaceutical
compositions featuring the compounds of the invention. The
compounds and compositions of the invention may also be used to
treat disorders where necroptosis is likely to play a substantial
role.
Inventors: |
Cuny; Gregory D.;
(Somerville, MA) ; Teng; Xin; (Framingham, MA)
; Yuan; Junying; (Newton, MA) ; Degterev;
Alexei; (Brookline, MA) ; Porco, JR.; John A.;
(Brookline, MA) |
Correspondence
Address: |
CLARK & ELBING LLP
101 FEDERAL STREET
BOSTON
MA
02110
US
|
Family ID: |
40351022 |
Appl. No.: |
12/228750 |
Filed: |
August 15, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60955966 |
Aug 15, 2007 |
|
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61038175 |
Mar 20, 2008 |
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Current U.S.
Class: |
514/361 ;
435/375; 514/423; 548/127; 548/537 |
Current CPC
Class: |
A61P 31/00 20180101;
A61P 35/00 20180101; C07D 277/56 20130101; C07D 417/12 20130101;
A61P 1/18 20180101; A61P 25/00 20180101; A61P 31/22 20180101; C07D
307/84 20130101; A61P 31/18 20180101; C07D 307/68 20130101; A61P
25/16 20180101; A61P 21/00 20180101; A61P 31/16 20180101; C07D
241/24 20130101; A61P 31/12 20180101; C07D 263/34 20130101; A61P
1/16 20180101; C07D 333/38 20130101; A61P 9/10 20180101; A61P 31/14
20180101; A61P 25/14 20180101; A61P 31/20 20180101; C07D 285/06
20130101; A61P 43/00 20180101; C07D 207/34 20130101; C07D 231/14
20130101; A61P 25/28 20180101 |
Class at
Publication: |
514/361 ;
548/127; 548/537; 514/423; 435/375 |
International
Class: |
A61K 31/433 20060101
A61K031/433; C07D 285/06 20060101 C07D285/06; C07D 207/30 20060101
C07D207/30; A61K 31/40 20060101 A61K031/40; C12N 5/00 20060101
C12N005/00; A61P 25/00 20060101 A61P025/00 |
Goverment Interests
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
[0002] The present research was supported by grants from the
National Institutes of Health (Grant No. GM-64703 and Grant No. U01
NS050560). The U.S. government has certain rights to this
invention.
Claims
1. A compound having a structure according to Formula (I)
##STR00098## wherein X.sub.1 and X.sub.2 are, independently, N or
CR.sup.4; X.sub.3 is selected from O, S, NR.sup.5, or
--(CR.sup.5).sub.2; Y is selected from C(O) or CH.sub.2; and Z is
(CR.sup.6R.sup.7).sub.n, R.sup.1 is selected from H, halogen,
optionally substituted C.sub.1-6 lower alkyl, or optionally
substituted C.sub.1-6 cycloalkyl, or optionally substituted aryl;
R.sup.2 is selected from H or optionally substituted C.sub.1-6
lower alkyl; R.sup.3 is optionally substituted aryl; each R.sup.4
is selected from H, halogen, carboxamido, nitro, cyano, optionally
substituted lower C.sub.1-6 alkyl, or optionally substituted aryl;
R.sup.5 is selected from H, halogen, optionally substituted lower
C.sub.1-6 alkyl, or optionally substituted aryl; each R.sup.6 and
R.sup.7 is, independently, selected from H or optionally
substituted C.sub.1-6 lower alkyl; and n is 0, 1, 2, or 3; wherein
when X.sub.1 and X.sub.2 are N, X.sub.3 is S, Y, is C(O), Z is
CH.sub.2, R.sup.2 is H, and R.sup.3 is 2-chloro-6-fluoro-phenyl,
R.sup.1 is not methyl; or any pharmaceutically acceptable salt or
solvate thereof, or stereoisomer thereof.
2. The compound of claim 1, wherein both X.sub.1 and X.sub.2 are N
or both X.sub.1 and X.sub.2 are CR.sup.4 and X.sub.3 is S or
NR.sup.5, or any pharmaceutically acceptable salt or solvate
thereof, or stereoisomer thereof.
3. The compound of claim 1, wherein said compound has a structure
according to Formula (I-a) ##STR00099## wherein R.sup.1, R.sup.2,
R.sup.3, R.sup.6, and R.sup.7 are as defined for Formula (I), or
any pharmaceutically acceptable salt or solvate thereof, or
stereoisomer thereof.
4. The compound of claim 3, wherein R.sup.3 is a substituted phenyl
group having the structure ##STR00100## wherein each R.sup.8,
R.sup.9, R.sup.10, R.sup.11, and R.sup.12 is selected,
independently, from H, lower C.sub.1-6 alkyl, halogen, amino,
carboxamido, alkoxy, nitro, and cyano, wherein at least one of
R.sup.8, R.sup.9, R.sup.10, R.sup.11, and R.sup.12 is not hydrogen,
or any pharmaceutically acceptable salt or solvate thereof, or
stereoisomer thereof.
5. The compound of claim 4, wherein R.sup.1 is C.sub.1-6 cycloalkyl
or branched C.sub.1-6 lower alkyl, R.sup.8 and R.sup.12 are
halogens, and R.sup.9, R.sup.10, and R.sup.11 are hydrogen, or any
pharmaceutically acceptable salt or solvate thereof, or
stereoisomer thereof.
6. The compound of claim 5, wherein R.sup.1 is cyclopropyl,
cyclobutyl, or isopropyl, R.sup.8 is fluorine, and R.sup.12 is
chlorine, or any pharmaceutically acceptable salt or solvate
thereof, or stereoisomer thereof.
7. The compound of claim 1, wherein R.sup.2 is H, or any
pharmaceutically acceptable salt or solvate thereof, or
stereoisomer thereof.
8. The compound of claim 1, wherein R.sup.6 and R.sup.7 are both
hydrogen, or any pharmaceutically acceptable salt or solvate
thereof, or stereoisomer thereof.
9. The compound of claim 1, wherein R.sup.6 is hydrogen and R.sup.7
is lower C.sub.1-6 alkyl, or any pharmaceutically acceptable salt
or solvate thereof, or stereoisomer thereof.
10. The compound of claim 9, wherein the carbon bearing R.sup.6 and
R.sup.7 has the (S)-configuration, or any pharmaceutically
acceptable salt or solvate thereof, or stereoisomer thereof.
11. The compound of claim 1, wherein said compound has a structure
according to Formula (I-b) ##STR00101## wherein Y and Z are as
defined for Formula (I) and R is selected from: hydrogen, halogen,
azido, cyano, nitro, optionally substituted lower C.sub.1-6 alkyl,
aryl, alkoxy, aryloxy, amino, carboxylic group, ketone, carbonate,
ester, carboxamide, or carbamate, or any pharmaceutically
acceptable salt or solvate thereof, or stereoisomer thereof.
12. The compound of claim 1, wherein said compound is
##STR00102##
13. The compound of claim 1, wherein said compound has a structure
according to Formula (I-c) ##STR00103## wherein R.sup.1, R.sup.2,
and R.sup.7 are as defined for Formula (I); R.sup.4A and R.sup.4B
are selected, independently, from hydrogen, halogen, carboxamido,
nitro, and cyano; R.sup.5 is H or optionally substituted C.sub.1-6
lower alkyl; each of R.sup.8, R.sup.9, R.sup.10, R.sup.11, and
R.sup.12 is selected, indepdently, from H, lower C.sub.1-6 alkyl,
halogen, amino, amido, alkoxy, nitro, and cyano, wherein at least
one of R.sup.8, R.sup.9, R.sup.10, R.sup.11, and R.sup.12 is not
hydrogen; or any pharmaceutically acceptable salt or solvate
thereof, or stereoisomer thereof.
14. The compound of claim 13, wherein said compound has a structure
according to Formula (I-d) ##STR00104## wherein R.sup.4B, R.sup.5,
R.sup.7, R.sup.8, R.sup.9, R.sup.10, R.sup.11, and R.sup.12 are as
defined in Formula (I-c), or any pharmaceutically acceptable salt
or solvate thereof, or stereoisomer thereof.
15. The compound of claim 14, wherein R.sup.8 and R.sup.12 are each
halogen and R.sup.9, R.sup.10, and R.sup.11 are hydrogen, or any
pharmaceutically acceptable salt or solvate thereof, or
stereoisomer thereof.
16. The compound of claim 13, wherein said compound has a structure
according to Formula (I-e) ##STR00105## wherein R.sup.1, R.sup.2,
R.sup.4A, R.sup.4B, R.sup.5, and R.sup.7 are as defined in Formula
(I-c), or any pharmaceutically acceptable salt or solvate thereof,
or stereoisomer thereof.
17. The compound of claim 16, wherein said compound is
##STR00106##
18. The compound of claim 13, wherein R.sup.7 is hydrogen, or any
pharmaceutically acceptable salt or solvate thereof, or
stereoisomer thereof.
19. The compound of claim 13, wherein R.sup.7 is optionally
substituted lower C.sub.1-6 alkyl, or any pharmaceutically
acceptable salt or solvate thereof, or stereoisomer thereof.
20. The compound of claim 13, wherein the carbon bearing R.sup.7
has the (S)-configuration, or any pharmaceutically acceptable salt
or solvate thereof, or stereoisomer thereof.
21. The compound of claim 1, selected from the group consisting of:
##STR00107## ##STR00108## ##STR00109## ##STR00110## ##STR00111##
##STR00112## ##STR00113## ##STR00114## ##STR00115## or any
pharmaceutically acceptable salt or solvate thereof, or
stereoisomer thereof.
22. A pharmaceutical composition comprising a pharmaceutically
acceptable excipient and the compound of claim 1, or the compound
having the formula: ##STR00116## or any pharmaceutically acceptable
salt or solvate thereof, or stereoisomer thereof.
23. A method of treating a condition in a subject, said method
comprising the step of administering the compound of claim 1, or
the compound having the formula: ##STR00117## or any
pharmaceutically acceptable salt or solvate thereof, or
stereoisomer thereof, to said subject in a dosage sufficient to
decrease necroptosis.
24. The method of claim 23, wherein said condition is a
neurodegenerative disease or is caused by alteration in cell
proliferation, differentiation, or intracellular signalling.
25. A method of decreasing necroptosis comprising contacting a cell
with the compound of claim 1, or the compound having the formula:
##STR00118## or any pharmaceutically acceptable salt or solvate
thereof, or stereoisomer thereof.
26. A kit comprising (a) a pharmaceutically acceptable composition
comprising the compound of claim 1, or the compound having the
formula ##STR00119## or any pharmaceutically acceptable salt or
solvate thereof, or stereoisomer thereof, and (b) instructions for
the use of the pharmaceutical composition of (a) to treat a
condition in a subject.
27. The method of claim 23, wherein said condition is stroke,
retinal or brain ischemic injury, head trauma, coronary heart
disease, or the result of cell death of cardiac muscle.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of U.S. Provisional
Application Nos. 60/955,966, filed Aug. 15, 2007, and 61/038,175,
filed Mar. 20, 2008, each of which is hereby incorporated by
reference.
FIELD OF THE INVENTION
[0003] The invention relates to heterocyclic compounds and to cell
death, in particular through necrosis and necroptosis, and
regulation thereof by heterocyclic compounds.
BACKGROUND OF THE INVENTION
[0004] In many diseases, cell death is mediated through apoptotic
and/or necrotic pathways. While much is known about the mechanisms
of action that control apoptosis, control of necrosis is not as
well understood. Understanding the mechanisms regulating both
necrosis and apoptosis in cells is essential to being able to treat
conditions, such as neurodegenerative diseases, stroke-coronary
heart disease, kidney disease, and liver disease. A thorough
understanding of necrotic and apoptotic cell death pathways is also
crucial to treating AIDS and the conditions associated with AIDS,
such as retinal necrosis.
[0005] Cell death has traditionally been categorized as either
apoptotic or necrotic based on morphological characteristics
(Wyllie et al., Int. Rev. Cytol. 68: 251 (1980)). These two modes
of cell death were also initially thought to occur via regulated
(caspase-dependent) and non-regulated processes, respectively. More
recent studies, however, demonstrate that the underlying cell death
mechanisms resulting in these two phenotypes are much more
complicated and under some circumstances interrelated. Furthermore,
conditions that lead to necrosis can occur by either regulated
caspase-independent or non-regulated processes.
[0006] One regulated caspase-independent cell death pathway with
morphological features resembling necrosis, called necroptosis, has
recently been described (Degterev et al., Nat. Chem. Biol. 1:112
(2005)). This manner of cell death can be initiated with various
stimuli (e.g., TNF-.alpha. and Fas ligand) and in an array of cell
types (e.g., monocytes, fibroblasts, lymphocytes, macrophages,
epithelial cells and neurons). Necroptosis may represent a
significant contributor to and in some cases predominant mode of
cellular demise under pathological conditions involving excessive
cell stress, rapid energy loss and massive oxidative species
generation, where the highly energy-dependent apoptosis process is
not operative.
[0007] The identification and optimization of low molecular weight
molecules capable of inhibiting necroptosis will assist in
elucidating its role in disease patho-physiology and could provide
compounds (i.e., necrostatins) for anti-necroptosis therapeutics.
The discovery of compounds that prevent caspase-independent cell
death (e.g., necrosis or necroptosis) would also provide useful
therapeutic agents for treating or preventing conditions in which
necrosis occurs. These compounds and methods would be particularly
useful for the treatment of neurodegenerative diseases, ischemic
brain and heart injuries, and head trauma.
SUMMARY OF THE INVENTION
[0008] In one aspect, the invention features a compound having a
structure according to Formula (I)
##STR00001##
wherein
[0009] X.sub.1 and X.sub.2 are, independently, N or CR.sup.4;
[0010] X.sub.3 is selected from O, S, NR.sup.5, or
--(CR.sup.5).sub.2;
[0011] Y is selected from C(O) or CH.sub.2; and
[0012] Z is (CR.sup.6R.sup.7).sub.n,
[0013] R.sup.1 is selected from H, halogen, optionally substituted
C.sub.1-6 lower alkyl, or optionally substituted C.sub.1-6
cycloalkyl, or optionally substituted aryl;
[0014] R.sup.2 is selected from H or optionally substituted
C.sub.1-6 lower alkyl;
[0015] R.sup.3 is optionally substituted aryl;
[0016] each R.sup.4 is selected from H, halogen, carboxamido,
nitro, cyano, optionally substituted lower C.sub.1-6 alkyl, or
optionally substituted aryl;
[0017] R.sup.5 is selected from H, halogen, optionally substituted
lower C.sub.1-6 alkyl, or optionally substituted aryl;
[0018] each R.sup.6 and R.sup.7 is, independently, selected from H,
optionally substituted aryl, or optionally substituted C.sub.1-6
lower alkyl; and
[0019] n is 0, 1, 2, or 3;
[0020] where
[0021] when X.sub.1 and X.sub.2 are N, X.sub.3 is S, Y, is C(O), Z
is CH.sub.2, R.sup.2 is H, and R.sup.3 is 2-chloro-6-fluoro-phenyl,
R.sup.1 is not methyl;
[0022] or any pharmaceutically acceptable salt or solvate thereof,
or stereoisomer thereof.
[0023] In some embodiments, both X.sub.1 and X.sub.2 are N or both
X.sub.1 and X.sub.2 are CR.sup.4, wherein X.sub.3 is not NR.sup.5
when X.sub.1 and X.sub.2 are N.
[0024] In some embodiments, X.sub.1 and X.sub.2 are N and X.sub.3
is S.
[0025] In some embodiments, X.sub.1 and X.sub.2 are CR.sup.4 and
X.sub.3 is NR.sup.5.
[0026] In some embodiments, R.sup.1 is C.sub.1-6 cycloalkyl or
branched C.sub.1-6 lower alkyl.
[0027] In some embodiments R.sup.2 is H.
[0028] In some embodiments, R.sup.3 is a substituted phenyl group
having the structure
##STR00002##
where
[0029] each R.sup.8, R.sup.9, R.sup.10, R.sup.11, and R.sup.12 is
selected, independently, from H, lower C.sub.1-6 alkyl, halogen,
amino, carboxamido, alkoxy, nitro, and cyano, and at least one of
R.sup.8, R.sup.9, R.sup.10, R.sup.11, and R.sup.12 is not
hydrogen.
[0030] In some embodiments, R.sup.8 and R.sup.12 are,
independently, halogen. In other embodiments, R.sup.8 and R.sup.12
are, independently, fluorine or chlorine. In certain embodiments,
R.sup.8 is fluorine and R.sup.12 is chlorine.
[0031] In some embodiments, R.sup.9, R.sup.10, and R.sup.11 are
hydrogen.
[0032] In some embodiments, R.sup.1 is C.sub.1-6 cycloalkyl or
branched C.sub.1-6 lower alkyl, or any pharmaceutically acceptable
salt or solvate thereof, or stereoisomer thereof. In other
embodiments, the compound of Formula (I) has a structure according
to Formula (I-a)
##STR00003##
[0033] where R.sup.1, R.sup.2, R.sup.3, R.sup.6, and R.sup.7 are as
defined for Formula (I), or any pharmaceutically acceptable salt or
solvate thereof, or stereoisomer thereof.
[0034] In some embodiments, R.sup.3 is a substituted phenyl group
having the structure
##STR00004##
where each R.sup.8, R.sup.9, R.sup.10, R.sup.11, and R.sup.12 is
selected, independently, from H, lower C.sub.1-6 alkyl, halogen,
amino, carboxamido, alkoxy, nitro, and cyano, and at least one of
R.sup.8, R.sup.9, R.sup.10, R.sup.11, and R.sup.12 is not
hydrogen.
[0035] In some embodiments, R.sup.8 and R.sup.12 are,
independently, halogen. In other embodiments, R.sup.8 and R.sup.12
are, independently, fluorine or chlorine. In certain embodiments,
R.sup.8 is fluorine and R.sup.12 is chlorine.
[0036] In some embodiments, R.sup.9, R.sup.10, and R.sup.11 are
hydrogen, or any pharmaceutically acceptable salt or solvate
thereof, or stereoisomer thereof.
[0037] In some embodiments, R.sup.1 is C.sub.1-6 cycloalkyl or
branched C.sub.16 lower alkyl, or any pharmaceutically acceptable
salt or solvate thereof, or stereoisomer thereof. In certain
embodiments, R.sup.1 is cyclopropyl, cyclobutyl, or isopropyl.
[0038] In some embodiments, R.sup.2 is H, or any pharmaceutically
acceptable salt or solvate thereof, or stereoisomer thereof.
[0039] In some embodiments, R.sup.6 and R.sup.7 are both hydrogen,
or any pharmaceutically acceptable salt or solvate thereof, or
stereoisomer thereof.
[0040] In some embodiments, R.sup.6 is hydrogen and R.sup.7 is
lower C.sub.1-6 alkyl, or any pharmaceutically acceptable salt or
solvate thereof, or stereoisomer thereof. In certain embodiments,
carbon bearing R.sup.6 and R.sup.7 has the (S)-configuration.
[0041] In other embodiments, the compound of Formula (I) has a
structure according to Formula (I-b)
##STR00005##
[0042] where Y and Z are as defined for Formula (I) and R is
selected from: hydrogen, halogen, azido, cyano, nitro, optionally
substituted lower C.sub.1-6 alkyl, aryl, alkoxy, aryloxy, amino,
carboxylic group, ketone, carbonate, ester, carboxamide, or
carbamate, or any pharmaceutically acceptable salt or solvate
thereof, or stereoisomer thereof.
[0043] In some embodiments, Y is C(O). In other embodiments, Y is
CH.sub.2.
[0044] In some embodiments, R is halogen. In certain embodiments, R
is chlorine, or any pharmaceutically acceptable salt or solvate
thereof, or stereoisomer thereof.
[0045] In some embodiments, Z is CH.sub.2. In other embodiments, Z
is CHR.sup.7, where R.sup.7 is C.sub.1-6 lower alkyl. In certain
embodiments, the carbon bearing R.sup.7 has the
S-configuration.
[0046] In some embodiments, the compound having a structure
according to (I-b) is
##STR00006##
[0047] or any pharmaceutically acceptable salt or solvate thereof,
or stereoisomer thereof.
[0048] In other embodiments, the compound of Formula (I) has a
structure according to Formula (I-c)
##STR00007##
[0049] wherein
[0050] R.sup.1, R.sup.2, and R.sup.7 are as defined for Formula
(I);
[0051] R.sup.4A and R.sup.4B are selected, independently, from
hydrogen, halogen, carboxamido, nitro, and cyano;
[0052] R.sup.5 is H or optionally substituted C.sub.1-6 lower
alkyl;
[0053] each of R.sup.8, R.sup.9, R.sup.10, R.sup.11, and R.sup.12
is selected, indepdently, from H, lower C.sub.1-6 alkyl, halogen,
amino, amido, alkoxy, nitro, and cyano, and at least one of
R.sup.8, R.sup.9, R.sup.10, R.sup.11, and R.sup.12 is not
hydrogen;
[0054] or any pharmaceutically acceptable salt or solvate thereof,
or stereoisomer thereof.
[0055] In some embodiments, R.sup.1 is H.
[0056] In some embodiments, R.sup.2 is H.
[0057] In some embodiments, R.sup.4A is H and R.sup.4B is CN. In
other embodiments, R.sup.4A is CN and R.sup.4B is H.
[0058] In some embodiments, R.sup.5 is unsubstituted C.sub.1-6
lower alkyl.
[0059] In some embodiments, R.sup.7 is C.sub.1-6 lower alkyl. In
other embodiments, the carbon bearing R.sup.7 has the
S-configuration.
[0060] In some embodiments, R.sup.8 and R.sup.12 are each,
independently, halogen.
[0061] In some embodiments, R.sup.9, R.sup.10, and R.sup.11 are
hydrogen.
[0062] In other embodiments, the compound of Formula (I) has a
structure according to Formula (I-d)
##STR00008##
[0063] wherein R.sup.4B, R.sup.5, R.sup.7, R.sup.8, R.sup.9,
R.sup.10, R.sup.11, and R.sup.12 are as defined in Formula (I-c),
or any pharmaceutically acceptable salt or solvate thereof, or
stereoisomer thereof.
[0064] In some embodiments, R.sup.4B is CN.
[0065] In some embodiments, R.sup.5 is unsubstituted C.sub.1-6
lower alkyl.
[0066] In some embodiments, R.sup.7 is C.sub.1-6 lower alkyl. In
certain embodiments, the carbon bearing R.sup.7 has the
S-configuration.
[0067] In some embodiments, R.sup.8 and R.sup.12 are each,
independently, halogen.
[0068] In some embodiments, R.sup.9, R.sup.10, and R.sup.11 are
hydrogen.
[0069] In other embodiments, the compound of Formula (I) has a
structure according to Formula (I-e)
##STR00009##
[0070] wherein R.sup.1, R.sup.2, R.sup.4A, R.sup.4B, R.sup.5, and
R.sup.7 are as defined in Formula (I-c), or any pharmaceutically
acceptable salt or solvate thereof, or stereoisomer thereof.
[0071] In some embodiments, R.sup.1 is H or unsubstituted C.sub.1-6
lower alkyl.
[0072] In some embodiments, R.sup.2 is H.
[0073] In some embodiments, R.sup.4A is H and R.sup.4B is CN.
[0074] In some embodiments, R.sup.4A is CN and R.sup.4B is H.
[0075] In some embodiments, R.sup.7 is hydrogen.
[0076] In some embodiments, R.sup.7 is C.sub.1-6 lower alkyl. In
certain embodiments, the carbon bearing R.sup.7 has the
S-configuration, or any pharmaceutically acceptable salt or solvate
thereof, or stereoisomer thereof.
[0077] In some embodiments, the compound of Formula (I-e) is
##STR00010##
[0078] In some embodiments, the compound of Formula (I) is selected
from the group consisting of:
##STR00011## ##STR00012## ##STR00013## ##STR00014## ##STR00015##
##STR00016## ##STR00017## ##STR00018##
or any pharmaceutically acceptable salt or solvate thereof, or
stereoisomer thereof.
[0079] The compounds of Formulas (I), (I-a), (I-b), (I-c), (I-d),
or (I-e) also include any pharmaceutically acceptable salts or
solvates thereof, or stereoisomers thereof.
[0080] In a second aspect, the invention features a pharmaceutical
composition including a pharmaceutically acceptable excipient and
the compound of any of Formulas (I), (I-a), (I-b), (I-c), (I-d), or
(I-e), or the compound having the formula:
##STR00019##
[0081] or any pharmaceutically acceptable salt or solvate thereof,
or stereoisomer thereof.
[0082] In a third aspect, the invention features a method of
treating a condition in a subject, where the method includes the
step of administering the compound of any of Formulas (I), (I-a),
(I-b), (I-c), (I-d), or (I-e), or the compound having the
formula:
##STR00020##
or any pharmaceutically acceptable salt or solvate thereof, or
stereoisomer thereof, to said subject in a dosage sufficient to
decrease necroptosis. In some embodiments, the condition is a
neurodegenerative disease or is caused by alteration in cell
proliferation, differentiation, or intracellular signalling.
[0083] In a fourth aspect, the invention features a method of
decreasing necroptosis including the step of contacting a cell with
the compound of any of Formulas (I), (I-a), (I-b), (I-c), (I-d), or
(I-e), or the compound having the formula:
##STR00021##
or any pharmaceutically acceptable salt or solvate thereof, or
stereoisomer thereof. In some embodiments, the condition is a
neurodegenerative disease or is a condition caused by alteration in
cell proliferation, differentiation, or intracellular signalling.
In some embodiments, the condition caused by alteration in cell
proliferation, differentiation, or intracellular signalling is
cancer or infection (e.g., by viruses (e.g., acute, latent and
persistent), bacteria, fungi, or other microbes). In some
embodiments, the viruses are human immunodeficiency virus (HIV),
Epstein-Barr virus (EBV), cytomegalovirus (CMV)5 human
herpesviruses (HHV), herpes simplex viruses (HSV), human T-Cell
leukemia viruses (HTLV)5 Varicella-Zoster virus (VZV), measles
virus, papovaviruses (JC and BK), hepatitis viruses, adenovirus,
parvoviruses, and human papillomaviruses. In other embodiments, the
condition is a neurodegenerative disease that is Alzheimer's
disease, Huntington's disease, Parkinson's disease, amyotrophic
lateral sclerosis, HIV-associated dementia, cerebral ischemia,
amyotropic lateral sclerosis, multiple sclerosis, Lewy body
disease, Menke's disease, Wilson's disease, Creutzfeldt-Jakob
disease, Fahr disease, or muscular dystrophies or related diseases
(e.g., Becker's muscular dystrophy, Duchenne muscular dystrophy,
myotonic dystrophy, limb-girdle muscular dystrophy,
Landouzy-Dejerine muscular dystrophy, facioscapulohumeral muscular
dystrophy (Steinert's disease), myotonia congenita, Thomsen's
disease, and Pompe's disease). In some embodiments, the
neurodegerative disease is muscle wasting. In other embodiments,
muscle wasting is associated with cancer, AIDS, congestive heart
failure, chronic obstructive pulmonary disease, and necrotizing
myopathy of intensive care.
[0084] In a fifth aspect, the invention features a method of
screening compounds to identify inhibitors of necroptosis, where
the method includes the following steps: [0085] (a) providing a
first cell in which necroptosis is inhibited; [0086] (b) contacting
the cell of (a) with a compound that inhibits necroptosis; [0087]
(c) comparing the inhibition of necroptosis observed in (b) to the
inhibition of necroptosis observed by contacting a cell with the
compound of any of Formulas (I), (I-a), (I-b), (I-c), (I-d), or
(I-e), or the compound having the formula:
[0087] ##STR00022## [0088] or any pharmaceutically acceptable salt
or solvate thereof, or stereoisomer thereof,
[0089] wherein an inhibitor of necroptosis is identified when the
inhibition of necroptosis observed in (b) exceeds the inhibition of
necroptosis by the compound of any of Formulas (I), (I-a), (I-b),
(I-c), (I-d), or (I-e), or any pharmaceutically acceptable salt or
solvate thereof, or stereoisomer thereof.
[0090] In a sixth aspect, the invention features a kit including
[0091] (a) a pharmaceutically acceptable composition that includes
the compound of any of Formulas (I), (I-a), (I-b), (I-c), (I-d), or
(I-e), or the compound having the formula:
[0091] ##STR00023## [0092] or any pharmaceutically acceptable salt
or solvate thereof, or stereoisomer thereof, and [0093] (b)
instructions for the use of the pharmaceutical composition of (a)
to treat a condition in a subject.
[0094] In another aspect, the compounds of Formulas (I), (I-a),
(I-b), (I-c), (I-d), or (I-e) are used to boost the immune system
in a patient. In some embodiments, the patient has an
immunocompromising condition. In other embodiments, the patient
does not have an immunocompromising condition.
[0095] By "alkoxy" is meant a group having the structure --O(lower
C.sub.1-6 alkyl), where the lower C.sub.1-6 alkyl may be brached,
linear, or cyclic. The lower C.sub.1-6 alkyl may also be
substituted or unsubstituted.
[0096] By "amino" is meant a group having a structure selected
from: --NH.sub.2, --NH(lower C.sub.1-6 alkyl), --N(lower C.sub.1-6
alkyl).sub.2, --NH(aryl), --N(lower C.sub.1-6 alkyl)(aryl), and
--N(aryl).sub.2. Each lower C.sub.1-6 alkyl and aryl may be,
independently, unsubstituted or substituted. Each lower C.sub.1-6
alkyl may be, independently, brached, linear, or cyclic.
[0097] By "aryl" is meant is an optionally substituted
C.sub.6-C.sub.14 cyclic group with [4n+2] .pi. electrons in
conjugation and where n is 1, 2, or 3. Non-limiting examples of
arenes include heteroaryls and benzene, naphthalene, anthracene,
and phenanthrene. Aryls may be unsubstituted or substituted. A
substituted aryl may be optionally substituted with 1, 2, 3, 4, 5,
or 6 substituents located at any position of the ring.
[0098] By "aryloxy" is meant a group having the structure
--O(aryl). Aryl may be unsubstituted or substituted.
[0099] By "azido" is meant a group having the structure
--N.sub.3.
[0100] By "carbamate" is meant a group having the structure
--OCONH.sub.2, --OCONH(lower C.sub.1-6 alkyl), --OCON(lower
C.sub.1-6 alkyl).sub.2, --OCON(lower C.sub.1-6 alkyl)(aryl),
--OCONH(aryl), or --OCON(aryl).sub.2. Each lower C.sub.1-6 alkyl
and aryl may be, independently, unsubstituted or substituted. Each
lower C.sub.1-6 alkyl may be, independently, brached, linear, or
cyclic.
[0101] By "carbonate" is meant a group having a the structure
--OCO.sub.2(lower C.sub.1-6 alkyl) or --OCO.sub.2(aryl). Each lower
C.sub.1-6 alkyl and aryl may be unsubstituted or substituted.
[0102] By "carboxamide" is meant a group having the structure
--CONH.sub.2, --CON(lower C.sub.1-6 alkyl), --CON(lower C.sub.1-6
alkyl).sub.2, --CON(lower C.sub.1-6 alkyl)(aryl), --CONH(aryl), or
--CON(aryl).sub.2. Each lower C.sub.1-6 alkyl and aryl may be,
independently, unsubstituted or substituted. Each lower C.sub.1-6
alkyl may be, independently, brached, linear, or cyclic.
[0103] By "carboxylic group" is meant a group having a structure
selected from: --CO.sub.2H, --CO.sub.2(lower C.sub.1-6 alkyl), and
--CO.sub.2(aryl). Each lower C.sub.1-6 alkyl and aryl may be
unsubstituted or substituted. Each lower C.sub.1-6 alkyl may be,
independently, brached, linear, or cyclic.
[0104] By "cyano" is meant a group having the structure --CN.
[0105] By "effective amount" or "therapeutically effective amount"
of an agent, as used herein, is that amount sufficient to effect
beneficial or desired results, such as clinical results, and, as
such, an effective amount depends upon the context in which it is
being applied. For example, in the context of administering an
agent that is an inhibitor of necroptosis, an effective amount of
an agent is, for example, an amount sufficient to achieve a
reduction in necroptosis as compared to the response obtained
without administration of the agent.
[0106] By "ester" is meant a group having a structure selected from
--OCO(lower C.sub.1-6 alkyl) or --OCO(aryl). Each lower C.sub.1-6
alkyl and aryl may be unsubstituted or substituted. Each lower
C.sub.1-6 alkyl may be, independently, brached, linear, or
cyclic.
[0107] By "halogen" or "halo" is meant fluorine (--F), chlorine
(--Cl), bromine (--Br), or iodine (--I).
[0108] By "heteroaryl" is mean an aryl group that contains 1, 2, or
3 heteroatoms in the cyclic framework. Exemplary heteroaryls
include, but are not limited to, furan, thiophene, pyrrole,
thiadiazole (e.g., 1,2,3-thiadiazole or 1,2,4-thiadiazole),
oxadiazole (e.g., 1,2,3-oxadiazole or 1,2,5-oxadiazole), oxazole,
benzoxazole, isoxazole, isothiazole, pyrazole, thiazole,
benzthiazole, triazole (e.g., 1,2,4-triazole or 1,2,3-triazole),
benzotriazole, pyridines, pyrimidines, pyrazines, quinoline,
isoquinoline, purine, pyrazine, pteridine, triazine (e.g,
1,2,3-triazine, 1,2,4-triazine, or 1,3,5-triazine)indoles,
1,2,4,5-tetrazine, benzo[b]thiophene, benzo[c]thiophene,
benzofuran, isobenzofuran, and benzimidazole. Heteroaryls may be
unsubstituted or substituted with 1, 2, 3, 4, 5, or 6
substitutents.
[0109] By "ketone" is meant a group having the structure --CO(lower
C.sub.1-6 alkyl) or --CO(aryl). Each lower C.sub.1-6 alkyl and aryl
may be unsubstituted or substituted. Each lower C.sub.1-6 alkyl may
be, independently, brached, linear, or cyclic.
[0110] By "lower alkyl" or "lower C.sub.1-6 alkyl" is meant
hydrocarbon chains of from 1 to 6 carbon atoms. Lower alkyls may
include 1, 2, 3, 4, 5, or 6 carbon atoms. A lower C.sub.1-6 alkyl
may be linear, branched or cyclic ("cycloalkyls"). Examples of
linear lower alkyl groups include, but are not limited to: methyl,
ethyl, n-propyl, n-butyl, n-pentyl, and n-hexyl. Examples of
branched lower alkyl groups include, but are not limited to:
isopropyl, s-, i- and t-butyl, and isoamyl. Examples of cyclic
lower alkyl groups include, but are not limited to: cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, and cyclobutylmethyl.
Desirably, a lower C.sub.1-6 alkyl is methyl, ethyl, propyl,
isopropyl, n-butyl, t-butyl, or cyclopropyl. A lower alkyl may be
unsubstituted or substituted. A substituted lower alkyl may be
optionally substituted with 1, 2, 3, 4, 5, or 6 substituents
located at any carbon of the lower alkyl.
[0111] By "nitro" is meant a group having the structure
--NO.sub.2.
[0112] A "pharmaceutically acceptable excipient" as used herein
refers any ingredient other than the compounds described herein
(for example, a vehicle capable of suspending or dissolving the
active compound) and having the properties of being nontoxic and
non-inflammatory in a patient. Excipients may include, for example:
antiadherents, antioxidants, binders, coatings, compression aids,
disintegrants, dyes (colors), emollients, emulsifiers, fillers
(diluents), film formers or coatings, flavors, fragrances, glidants
(flow enhancers), lubricants, preservatives, printing inks,
sorbents, suspensing or dispersing agents, sweeteners, or waters of
hydration. Exemplary excipients include, but are not limited to:
butylated hydroxytoluene (BHT), calcium carbonate, calcium
phosphate (dibasic), calcium stearate, croscarmellose, crosslinked
polyvinyl pyrrolidone, citric acid, crospovidone, cysteine,
ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl
methylcellulose, lactose, magnesium stearate, maltitol, mannitol,
methionine, methylcellulose, methyl paraben, microcrystalline
cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone,
pregelatinized starch, propyl paraben, retinyl palmitate, shellac,
silicon dioxide, sodium carboxymethyl cellulose, sodium citrate,
sodium starch glycolate, sorbitol, starch (corn), stearic acid,
stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin
E, vitamin C, and xylitol.
[0113] The term "pharmaceutically acceptable salt," as used herein,
represents those salts which are, within the scope of sound medical
judgment, suitable for use in contact with the tissues of humans
and animals without undue toxicity, irritation, allergic response
and the like and are commensurate with a reasonable benefit/risk
ratio. Pharmaceutically acceptable salts are well known in the art.
For example, S. M. Berge et al. describe pharmaceutically
acceptable salts in detail in J. Pharmaceutical Sciences, 1977,
66:1-19. The salts can be prepared in situ during the final
isolation and purification of the compounds of the invention or
separately by reacting the free base group with a suitable organic
acid. Representative acid addition salts include acetate, adipate,
alginate, ascorbate, aspartate, benzenesulfonate, benzoate,
bisulfate, borate, butyrate, camphorate, camphersulfonate, citrate,
cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, fumarate, glucoheptonate, glycerophosphate,
hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride,
hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate,
laurate, lauryl sulfate, malate, maleate, malonate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,
oleate, oxalate, palmitate, pamoate, pectinate, persulfate,
3-phenylpropionate, phosphate, picrate, pivalate, propionate,
stearate, succinate, sulfate, tartrate, thiocyanate,
toluenesulfonate, undecanoate, valerate salts and the like.
Representative alkali or alkaline earth metal salts include sodium,
lithium, potassium, calcium, magnesium and the like, as well as
nontoxic ammonium, quaternary ammonium, and amine cations,
including, but not limited to ammonium, tetramethylammonium,
tetraethylammonium, methylamine, dimethylamine, trimethylamine,
triethylamine, ethylamine and the like.
[0114] The term "pharmaceutically acceptable solvates," as used
herein, refers to compounds that retain non-covalent associations
to residual solvent molecules in the solid state. For example,
solvates may be prepared by crystallization, recrystallization, or
precipitation from a solution that includes organic solvents,
water, or a mixture thereof. Solvates include, but are not limited
to, compounds that include solvent molecules in the crystal lattice
following recrystallization. The molecular stoichiometry of
solvation can vary from, for example, 1:1 solvent:compound to 10:1
solvent:compound. These ratios can include a mixture of associated
solvent molecules. Exemplary, non-limiting examples of solvents
that can form solvates with the compounds of the invention include
water (for example, mono-, di-, and tri-hydrates),
N-methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO),
N,N'-dimethylformamide (DMF), N,N'-dimethylacetamide (DMAC),
1,3-dimethyl-2-imidazolidinone (DMEU),
1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU),
acetonitrile (ACN), propylene glycol, ethyl acetate, benzyl
alcohol, 2-pyrrolidone, benzyl benzoate, or any combination
thereof.
[0115] By "pharmaceutical composition" is meant a composition
containing a compound of the invention, formulated with a
pharmaceutically acceptable excipient, and manufactured or sold
with the approval of a governmental regulatory agency as part of a
therapeutic regimen for the treatment of disease in a mammal.
Excipients consisting of DMSO are specifically excluded.
Pharmaceutical compositions can be formulated, for example, for
oral administration in unit dosage form (e.g., a tablet, capsule,
caplet, gelcap, or syrup); for topical administration (e.g., as a
cream, gel, lotion, or ointment); for intravenous administration
(e.g., as a sterile solution free of particulate emboli and in a
solvent system suitable for intravenous use); or any other
formulation described herein.
[0116] By "stereoisomer" is meant a diastereomer, enantiomer, or
epimer of a compound. A chiral center in a compound may have the
S-configuration or the R-configuration. Diastereomers of a compound
include stereoisomers in which some, but not all, of the chiral
centers have the opposite configuration as well as those compounds
in which substituents are differently oriented in space (for
example, trans versus cis).
[0117] Where a group is substituted, the group may be substituted
with 1, 2, 3, 4, 5, or 6 substituents. Optional substituents
include, but are not limited to: halogen, azido, cyano, nitro,
lower C.sub.1-6 alkyl, aryl, alkoxy, aryloxy, amino, carboxylic
group, ketone, carbonate, ester, carboxamide, or carbamate.
Substituents may be further substituted with 1, 2, 3, 4, 5, or 6
substituents as defined herein. For example, a lower C.sub.1-6
alkyl or an aryl group (e.g., heteroaryl, phenyl, or naphthyl) may
be further substituted with 1, 2, 3, 4, 5, or 6 substituents.
BRIEF DESCRIPTION OF THE DRAWINGS
[0118] FIG. 1 shows cell-type-specific activities of necrostatins.
FADD-deficient Jurkat, L929 and mouse adult lung fibroblast cells
were treated for 24 hours with 10 ng/mL human TNF-.alpha. and/or
100 .mu.M zVAD.fmk as indicated in the presence of 30 .mu.M of
necrostatin 1, 2 or 55. Cell viability was determined using a
commercial cell viability kit. Values were normalized to cells
treated with necrostatins in the absence of necroptotic stimulus,
which were set as 100% viability. Error bars reflect standard
deviation values (N=2).
DETAILED DESCRIPTION OF THE INVENTION
[0119] We have discovered a series of heterocyclic derivatives that
inhibit tumor necrosis factor alpha (TNF-.alpha.)-induced
necroptosis. The heterocyclic compounds of the invention are
described by Formulas (I) and (Ia)-(Ie) and are shown to inhibit
TNF-.alpha. induced necroptosis in FADD-deficient variant of human
Jurkat T cells. Pharmaceutical compositions including the compounds
of the invention are also described. The invention also features
kits and methods of treatment featuring the compounds and
compositions of the invention.
[0120] Compounds of the invention are described generally by
Formula (I):
##STR00024##
wherein
[0121] X.sub.1 and X.sub.2 are, independently, N or CR.sup.4;
[0122] X.sub.3 is selected from O, S, NR.sup.5, or
--(CR.sup.5).sub.2;
[0123] Y is selected from C(O) or CH.sub.2; and
[0124] Z is (CR.sup.6R.sup.7).sub.n,
[0125] R.sup.1 is selected from H, halogen, optionally substituted
C.sub.1-6 lower alkyl, or optionally substituted C.sub.1-6
cycloalkyl;
[0126] R.sup.2 is selected from H or optionally substituted
C.sub.1-6 lower alkyl;
[0127] R.sup.3 is optionally substituted aryl;
[0128] each R.sup.4 is selected from H, halogen, optionally
substituted lower C.sub.1-6 alkyl, or optionally substituted
aryl;
[0129] R.sup.5 is selected from H, halogen, optionally substituted
lower C.sub.1-6 alkyl, or optionally substituted aryl;
[0130] each R.sup.6 and R.sup.7 is, independently, selected from H,
optionally substituted aryl, or optionally substituted C.sub.16
lower alkyl; and
[0131] n is 0, 1, 2, or 3; where
[0132] when X.sub.1 and X.sub.2 are N, X.sub.3 is S, Y, is C(O), Z
is CH.sub.2, R.sup.2 is H, and R.sup.3 is 2-chloro-6-fluoro-phenyl,
R.sup.1 is not methyl;
[0133] or any pharmaceutically acceptable salt or solvate thereof,
or stereoisomer thereof.
[0134] In one embodiment, the compounds of the invention have the
Formula (I-a):
##STR00025##
[0135] where R.sup.1 is selected from H, halogen, optionally
substituted C.sub.1-6 lower alkyl, optionally substituted C.sub.1-6
cycloalkyl, or optionally substituted aryl;
[0136] R.sup.2 is selected from H or optionally substituted
C.sub.1-6 lower alkyl;
[0137] R.sup.3 is optionally substituted aryl;
[0138] R.sup.6 and R.sup.7 are, independently, selected from H or
optionally substituted C.sub.1-6 lower alkyl; and
[0139] at least one of R.sup.6 and R.sup.7 is hydrogen.
[0140] Compounds of the invention having Formula (I-a) include:
TABLE-US-00001 ##STR00026## ##STR00027## ##STR00028## ##STR00029##
##STR00030## ##STR00031## ##STR00032## ##STR00033## ##STR00034##
##STR00035## ##STR00036## ##STR00037## ##STR00038## ##STR00039##
##STR00040## ##STR00041## ##STR00042## ##STR00043## ##STR00044##
##STR00045## ##STR00046## ##STR00047## ##STR00048## ##STR00049##
##STR00050## ##STR00051##
[0141] Compounds of the invention also include those having Formula
(I-b):
##STR00052##
[0142] wherein Y and Z are as defined for Formula (I) and R is a
substituent that may be selected from: hydrogen, halogen, azido,
cyano, nitro, optionally substituted lower C.sub.1-6 alkyl, aryl,
alkoxy, aryloxy, amino, carboxylic group, ketone, carbonate, ester,
carboxamide, or carbamate.
[0143] Compounds of the invention having Formula (I-b) include:
TABLE-US-00002 ##STR00053## ##STR00054## ##STR00055## ##STR00056##
##STR00057## ##STR00058## ##STR00059## ##STR00060## ##STR00061##
##STR00062##
[0144] Compounds of the invention can also have a structure
according to Formula (I-c):
##STR00063##
where
[0145] R.sup.1 is selected from H, halogen, optionally substituted
C.sub.1-6 lower alkyl, optionally substituted C.sub.1-6 cycloalkyl,
or optionally substituted aryl;
[0146] R.sup.2 is selected from H or optionally substituted
C.sub.1-6 lower alkyl;
[0147] R.sup.4A and R.sup.4B are selected, independently, from
hydrogen, halogen, carboxamido, nitro, and cyano;
[0148] R.sup.5 is H or optionally substituted C.sub.1-6 lower
alkyl;
[0149] R.sup.7 is hydrogen or optionally substituted lower
C.sub.1-6 alkyl;
[0150] each of R.sup.8, R.sup.9, R.sup.10, R.sup.11, and R.sup.12
is selected, indepdently, from H, lower C.sub.1-6 alkyl, halogen,
amino, amido, alkoxy, nitro, and cyano; and
[0151] at least one of R.sup.8, R.sup.9, R.sup.10, R.sup.11, and
R.sup.12 is not hydrogen.
[0152] Examples of compounds having Formula (I-c) include those
having Formula (I-d):
##STR00064##
[0153] where R.sup.4B, R.sup.5, R.sup.7, R.sup.8, R.sup.9,
R.sup.10, R.sup.11, and R.sup.12 are as defined in Formula
(I-c).
[0154] Additional examples of compounds having Formula (I-c)
include those having Formula (I-e):
##STR00065##
[0155] where R.sup.1, R.sup.2, R.sup.4A, R.sup.4B, R.sup.5, and
R.sup.7 are as defined in Formula (I-c).
[0156] Exemplary compounds of the invention having Formula (I-e)
include those depicted in Table 1:
TABLE-US-00003 TABLE 1 R.sup.1 R.sup.2 R.sup.4A R.sup.4B R.sup.5
R.sup.7 Structure 78 H H H Cl Me H ##STR00066## 80 H H H H H H
##STR00067## 81 Me H H H H H ##STR00068## 82 Me H H H Me H
##STR00069## 83 Me H H H i-Pr H ##STR00070## 84 H H H H Me H
##STR00071## 85 H H H H i-Pr H ##STR00072## 86 H H H H Bn H
##STR00073## 87 H H H H CH(Me)Ph H ##STR00074## 88 H Me H H Me H
##STR00075## 89 H H H H i-Pr (.+-.)-Me ##STR00076## 90 H H H H i-Pr
(S)-Me ##STR00077## 91 H H H H i-Pr (R)-Me ##STR00078## 92 H H H H
i-Pr (.+-.)-n-Bu ##STR00079## 93 H H H Br Et H ##STR00080## 94 H H
H CN Me H ##STR00081## 95 H H H CN Et H ##STR00082## 96 H H H CN
i-Pr H ##STR00083## 97 H H H CN Me (S)-Me ##STR00084## 98 H H CN H
Me H ##STR00085## 99 H H CN H i-Pr H ##STR00086##
[0157] Compounds of the invention where the carbon bearing R.sup.7
is a chiral center may be used as a racemate, stereochemical
mixture, or in enantiomerically pure form. In one embodiment, a
compound where the carbon bearing R.sup.7 is a chiral center has
the (S)-configuration. In another embodiment, a compound where the
carbon bearing R.sup.7 is a chiral center has the
(R)-configuration.
[0158] Compounds of the invention can be synthesized according to
methods known in the art or by the methods provided in the examples
below. For example, compounds of the invention (e.g., compounds of
Formula (I-b)) may be prepared according to the method illustrated
in Scheme 1 and presented in Example 1.
##STR00087##
Pharmaceutical Compositions
[0159] The compounds of the invention (e.g., (Formulas (I) and
(I-a)-(I-e)) can be formulated into pharmaceutical compositions for
administration to human subjects in a biologically compatible form
suitable for administration in vivo. Accordingly, in another
aspect, the present invention provides a pharmaceutical composition
comprising a compound of the invention in admixture with a
pharmaceutically acceptable excipient. Conventional procedures and
ingredients for the selection and preparation of suitable
formulations are described, for example, in Remington's
Pharmaceutical Sciences (2003-20.sup.th edition) and in The United
States Pharmacopeia: The National Formulary (USP 24 NF 19),
published in 1999.
[0160] The compounds of the invention may be used in the form of
the free base, in the form of salts, solvates, and as prodrugs. All
forms are within the scope of the invention. In accordance with the
methods of the invention, the described compounds or salts,
solvates, or prodrugs thereof may be administered to a patient in a
variety of forms depending on the selected route of administration,
as will be understood by those skilled in the art. The compounds of
the invention may be administered, for example, by oral,
parenteral, buccal, sublingual, nasal, rectal, patch, pump, or
transdermal administration and the pharmaceutical compositions
formulated accordingly. Parenteral administration includes
intravenous, intraperitoneal, subcutaneous, intramuscular,
transepithelial, nasal, intrapulmonary, intrathecal, rectal, and
topical modes of administration. Parenteral administration may be
by continuous infusion over a selected period of time.
Pharmaceutically Acceptable Excipients
[0161] Pharmaceutically acceptable excipients may include, for
example: antiadherents, antioxidants, binders, coatings,
compression aids, disintegrants, dyes (colors), emollients,
emulsifiers, fillers (diluents), film formers or coatings, flavors,
fragrances, glidants (flow enhancers), lubricants, preservatives,
printing inks, sorbents, suspensing or dispersing agents,
sweeteners, or waters of hydration. Exemplary excipients include,
but are not limited to: butylated hydroxytoluene (BHT), calcium
carbonate, calcium phosphate (dibasic), calcium stearate,
croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid,
crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl
cellulose, hydroxypropyl methylcellulose, lactose, magnesium
stearate, maltitol, mannitol, methionine, methylcellulose, methyl
paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl
pyrrolidone, povidone, pregelatinized starch, propyl paraben,
retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl
cellulose, sodium citrate, sodium starch glycolate, sorbitol,
starch (corn), stearic acid, stearic acid, sucrose, talc, titanium
dioxide, vitamin A, vitamin E, vitamin C, and xylitol.
Oral Administration
[0162] A compound of the invention may be orally administered, for
example, with an inert diluent or with an assimilable edible
carrier, or it may be enclosed in hard or soft shell gelatin
capsules, or it may be compressed into tablets, or it may be
incorporated directly with the food of the diet. For oral
therapeutic administration, a compound of the invention may be
incorporated with an excipient and used in the form of ingestible
tablets, buccal tablets, troches, capsules, elixirs, suspensions,
syrups, wafers, and the like.
Parenteral Administration
[0163] A compound of the invention may also be administered
parenterally. The pharmaceutical forms suitable for injectable use
include sterile aqueous solutions or dispersions and sterile
powders for the extemporaneous preparation of sterile injectable
solutions or dispersions. In all cases the form must be sterile and
must be fluid to the extent that may be easily administered via
syringe.
Nasal Administration
[0164] Compositions for nasal administration may conveniently be
formulated as aerosols, drops, gels, and powders. Aerosol
formulations typically include a solution or fine suspension of the
active substance in a physiologically acceptable aqueous or
non-aqueous solvent and are usually presented in single or
multidose quantities in sterile form in a sealed container, which
can take the form of a cartridge or refill for use with an
atomizing device. Alternatively, the sealed container may be a
unitary dispensing device, such as a single dose nasal inhaler or
an aerosol dispenser fitted with a metering valve which is intended
for disposal after use. Where the dosage form comprises an aerosol
dispenser, it will contain a propellant, which can be a compressed
gas, such as compressed air or an organic propellant, such as
fluorochlorohydrocarbon. The aerosol dosage forms can also take the
form of a pump-atomizer.
Buccal or Sublingual Administration
[0165] Compositions suitable for buccal or sublingual
administration include tablets, lozenges, and pastilles, where the
active ingredient is formulated with a carrier, such as sugar,
acacia, tragacanth, or gelatin and glycerine. Compositions for
rectal administration are conveniently in the form of suppositories
containing a conventional suppository base, such as cocoa
butter.
[0166] The compounds of the invention may be administered to an
animal alone or in combination with pharmaceutically acceptable
carriers, as noted above, the proportion of which is determined by
the solubility and chemical nature of the compound, chosen route of
administration, and standard pharmaceutical practice.
Dosage Amounts
[0167] The amount of active ingredient in the compositions of the
invention can be varied. One skilled in the art will appreciate
that the exact individual dosages may be adjusted somewhat
depending upon a variety of factors, including the protein being
administered, the time of administration, the route of
administration, the nature of the formulation, the rate of
excretion, the nature of the subject's conditions, and the age,
weight, health, and gender of the patient. Generally, dosage levels
of between 0.1 .mu.g/kg to 100 mg/kg of body weight are
administered daily as a single dose or divided into multiple doses.
Desirably, the general dosage range is between 250 .mu.g/kg to 5.0
mg/kg of body weight per day. Wide variations in the needed dosage
are to be expected in view of the differing efficiencies of the
various routes of administration. For instance, oral administration
generally would be expected to require higher dosage levels than
administration by intravenous injection. Variations in these dosage
levels can be adjusted using standard empirical routines for
optimization, which are well known in the art. In general, the
precise therapeutically effective dosage will be determined by the
attending physician in consideration of the above identified
factors.
Therapeutic Uses and Screening Methods
[0168] Compounds disclosed herein can be used to treat disorders
where necroptosis is likely to play a substantial role (e.g.,
cerebral ischemia, traumatic brain injury, and other disorders
described herein). Compounds of the invention can also be used in
screening methods to identify targets of necroptosis and to
identify additional inhibitors of necroptosis, as well as in assay
development.
[0169] Compounds disclosed herein can be evaluated for their
pharmacological properties in animal models of disease. The
compounds identified to decrease necrosis or necroptosis may be
structurally modified and subsequently used to decrease necrosis or
necroptosis, or to treat a subject with a condition in which
necrosis or necroptosis occurs. The methods used to generate
structural derivatives of the small molecules that decrease
necrosis or necroptosis are readily known to those skilled in the
fields of organic and medicinal chemistry.
[0170] Therapy according to the invention may be performed alone or
in conjunction with another therapy, for example in combination
with apoptosis inhibitors, and may be provided at home, the
doctor's office, a clinic, a hospital's outpatient department, or a
hospital. Treatment generally begins at a hospital so that the
doctor can observe the therapy's effects closely and make any
adjustments that are needed. The duration of the therapy depends on
the age and condition of the patient, as well as how the patient
responds to the treatment. Additionally, a person having a greater
risk of developing a condition may receive prophylactic treatment
to inhibit or delay symptoms of the disease.
[0171] In some embodiments, the compounds and methods of the
invention can be used to treat any of the following disorders where
necroptosis is likely to play a substantial role: a
neurodegenerative disease of the central or peripheral nervous
system, the result of retinal neuronal cell death, the result of
cell death of cardiac muscle, the result of cell death of cells of
the immune system; stroke, liver disease, pancreatic disease, the
result of cell death associated with renal failure; heart,
mesenteric, retinal, hepatic or brain ischemic injury, ischemic
injury during organ storage, head trauma, septic shock, coronary
heart disease, cardiomyopathy, bone avascular necrosis, sickle cell
disease, muscle wasting, gastrointestinal disease, tuberculosis,
diabetes, alteration of blood vessels, muscular dystrophy,
graft-versus-host disease, viral infection, Crohn's disease,
ulcerative colitis, asthma, and any condition in which alteration
in cell proliferation, differentiation or intracellular signaling
is a causative factor.
Conditions Caused by Alteration in Cell Proliferation,
Differentiation, or Intracellular Signalling
[0172] Conditions in which alteration in cell proliferation,
differentiation or intracellular signaling is a causative factor
include cancer and infection, e.g., by viruses (e.g., acute, latent
and persistent), bacteria, fungi, or other microbes. Exemplary
viruses are human immunodeficiency virus (HIV), Epstein-Barr virus
(EBV), cytomegalovirus (CMV)5 human herpesviruses (HHV), herpes
simplex viruses (HSV), human T-Cell leukemia viruses (HTLV)5
Varicella-Zoster virus (VZV), measles virus, papovaviruses (JC and
BK), hepatitis viruses, adenovirus, parvoviruses, and human
papillomaviruses.
Neurodegenerative Diseases
[0173] Exemplary neurodegenerative diseases are Alzheimer's
disease, Huntington's disease, Parkinson's disease, amyotrophic
lateral sclerosis, HIV-associated dementia, cerebral ischemia,
amyotropic lateral sclerosis, multiple sclerosis, Lewy body
disease, Menke's disease, Wilson's disease, Creutzfeldt-Jakob
disease, and Fahr disease. Exemplary muscular dystrophies or
related diseases are Becker's muscular dystrophy, Duchenne muscular
dystrophy, myotonic dystrophy, limb-girdle muscular dystrophy,
Landouzy-Dejerine muscular dystrophy, facioscapulohumeral muscular
dystrophy (Steinert's disease), myotonia congenita, Thomsen's
disease, and Pompe's disease. Muscle wasting can be associated with
cancer, AIDS, congestive heart failure, and chronic obstructive
pulmonary disease, as well as include necrotizing myopathy of
intensive care.
[0174] Compounds and methods of the invention can additionally be
used to boost the immune system, whether or not the patient being
treated has an immunocompromising condition. For example, the
compounds described herein can be used in a method to strengthen
the immune system during immunization, e.g., by functioning as an
adjuvant, or by being combined with an adjuvant.
Kits
[0175] Any of the compounds or pharmaceutical compositions of the
invention can be used together with a set of instructions, i.e., to
form a kit. The kit may include instructions for use of the
compounds of the invention in a screening method or as a therapy as
described herein.
[0176] The following non-limiting examples are illustrative of the
present invention.
EXAMPLES
Example 1
Preparation of [1,2,3]thiadiazole derivatives of Formula (I-a)
[0177] The [1,2,3]thiadiazole derivatives are prepared according to
the method outlined in Scheme 1. Meldrum's acid was treated with
acyl chlorides in the presence of pyridine to give .beta.-ketoester
(step (a); Oikawa et al., J. Org. Chem. 43: 2087 (1978)). The
esters were allowed to react with mono-Boc-hydrazine in the
presence of a catalytic amount of p-toluenesulfonic acid (p-TsOH)
to give the corresponding imines (step (b); Thomas et al., J. Med.
Chem. 28: 442 (1985)). Cyclization in the presence of thionyl
chloride yielded the [1,2,3]thiadiazole esters (step (c)). Base
hydrolysis of the esters provided acids (step (d)). These materials
were coupled with various amines utilizing HBTU (Method A), the
corresponding acyl chlorides (Method B) or through the use of EDCI
(Method C) to give amides of Formula (I-b).
Example 2
Preparation of Compounds (13) and (16)
##STR00088##
[0179] Compound 13 was prepared according to the procedure outlined
in Scheme 2. The ester was reduced with sodium borohydride (step
(a)) and the product alcohol was converted to the corresponding
aldehyde utilizing Dess-Martin reagent (step (b)). The aldehyde was
condensed with 2-chloro-6-fluorobenzylamine in the presence of
anhydrous magnesium sulfate to give an imine, which was
subsequently reduced with sodium triacetoxyborohydride to give the
secondary amine 13 (step (c)). The imide derivative 16 was also
prepared starting with a carboxylic acid which was first converted
to the corresponding acid chloride (step (d)). This material was
then allowed to react with the anion of 2-chloro-6-fluorobenzamide
generated with sodium hydride to give imide 16 in 34% yield (step
(e)).
Example 3
Preparation of .alpha.-substituted
(.+-.)-2-chloro-6-fluorobenzylamines
##STR00089##
[0181] The .alpha.-substituted (.+-.)-2-chloro-6-fluorobenzylamines
were prepared according to Scheme 3 (Polniaszek et al., J. Org.
Chem., 55: 215 (1990)). 2-Chloro-6-fluorophenyl ketones were
reduced with borane-tetrahydrofuran complex to give the secondary
alcohols (step (a)). The alcohols were converted to the
corresponding phthalimides via a Mitsonobu reaction (step (b)). The
benzylamines were isolated following treatment with hydrazine
monohydrate (step (c)). (S)-1-(2-Chloro-6-fluorophenyl)ethylamine
was prepared by treating the benzonitrile starting material with
methyl magnesium chloride followed by treatment with acetic
anhydride to give .alpha.-enamide (step (d)). Asymmetric
hydrogenation in the presence of the chiral catalyst
(S,S)-Me-BPE-Rh gave the corresponding amide (step (e); Burk et
al., J. Am. Chem. Soc. 118: 5142 (1996)). Acid hydrolysis of the
amide yielded the optically pure amine (step (f)), isolated as the
hydrochloride salt.
Example 4
Synthesis of 3-Alkyl pyrrole derivatives (Compounds (81), (82), and
(83))
##STR00090##
[0183] 3-Alkyl pyrrole derivatives were prepared according to the
procedure outlined in Scheme 1. Glycine ethyl ester, 5, was treated
with p-toluenesulfonyl chloride (Ts--Cl) to give 6, which upon
treatment with 4-diethylaminobutan-2-one in the presence of t-BuOK
gave 7. Dehydration with POCl.sub.3 yielded the dihydropyrrole
derivative 8. Elimination in the presence of sodium ethoxide
generated pyrrole derivative 9. The pyrrole nitrogen was
deprotonated with sodium hydride and alkylated to give 10. The
ester was hydrolyzed with aqueous KOH in MeOH and then the
corresponding acid 11 was converted to amides (81), (82), and (83)
using EDCI.
Example 5
Synthesis of 1-Alkyl pyrrole derivatives
##STR00091##
[0185] 1-Alkyl pyrrole derivatives were prepared according to the
procedure outlined in Scheme 2. Methyl 2-pyrrolecarboxylate, 100,
was deprotonated using sodium hydride and then alkylated to give
101. The ester was hydrolyzed to give acid 102, which was coupled
to a 2-chloro-6-fluorobenzylamine utilizing EDCI to give amide 103.
Regioselective bromination with NBS gave 104. Finally, conversion
of the aryl bromide to a nitrile was accomplished utilizing a
palladium-mediated coupling with zinc cyanide to give 105 in
excellent yield.
Example 6
Synthesis of cyano- and halo-substituted pyrrole derivatives
##STR00092##
[0187] Cyano- and halo-substituted pyrrole derivatives were
prepared according to the procedure outlined in Scheme 3.
1-Alkylpyrroles 101 were allowed to react with chlorosulfonyl
isocyanate to give two readily separable regioisomeric cyanopyrrole
derivatives 106 and 107 (1:4). Each was converted the corresponding
acid and then coupled with 2-chloro-6-fluorobenzylamine to give 108
and 109, respectively. Methyl 2-pyrrolecarboxylate, 100, was also
regioselectively chlorinated with t-butyl hypochlorite to give 110.
N-alkylation gave 111 and subsequent ester hydrolysis yielded 112,
which was coupled with 2-chloro-6-fluorobenzylamine to give
compound (78).
Example 7
Evaluation of Necroptosis Inhibitory Activity by Thiadiazoles
[0188] Evaluation of necroptosis inhibitory activity was performed
using a FADD-deficient variant of human Jurkat T cells treated with
TNF-.alpha. as previously described (Degterev et al., Nat. Chem.
Biol. 1:112 (2005) and Jagtap et al., J. Med. Chem. 50: 1886
(2007)). Utilizing these conditions the cells efficiently underwent
necroptosis, which was completely and selectively inhibited by 1
(see Scheme 7; EC.sub.50=0.050 .mu.M). For EC.sub.50 value
determinations, cells were treated with 10 ng/mL of human
TNF-.alpha. in the presence of increasing concentration of test
compounds for 24 hours followed by ATP-based viability
assessment.
[0189] ATP-based viability assessment: Briefly, necroptosis
activity was performed using a FADD-deficient variant of human
Jurkat T cells treated with TNF-.alpha.. For EC.sub.50 value
determinations, cells (500,000 cells/mL, 100 .mu.L per well in a
96-well plate) were treated with 10 ng/mL of human TNF-.alpha. in
the presence of increasing concentration of test compounds for 24
hours at 37.degree. C. in a humidified incubator with 5% CO.sub.2
followed by ATP-based viability assessment. Stock solutions (30 mM)
in DMSO were initially prepared and then diluted with DMSO to give
testing solutions, which were added to each test well. The final
DMSO concentration was 0.5%. Eleven compound test concentrations
(0.030-100 .mu.M) were used. Each concentration was done in
duplicate.
[0190] Cell viability assessments were performed using a commercial
luminescent ATP-based assay kit (CellTiter-Glo, Promega, Madison,
Wis.) according to the manufacturer's instructions. Briefly, 40
.mu.L of the cell lysis/ATP detection reagent was added to each
well. Plates were incubated on a rocking platform for 10 minutes at
room temperature and luminescence was measured using a Wallac
Victor 3 plate-reader (Perkin Elmer, Wellesley, Mass.). Cell
viability was expressed as a ratio of the signal in the well
treated with TNF-.alpha. and compound to the signal in the well
treated with compound alone. This was done to account for
nonspecific toxicity, which in most cases was <10%. EC.sub.50
values were calculated using nonlinear regression analysis of
sigmoid dose-response (variable slope) curves from plots of log[I]
verses viability values.
[0191] Table 2 provides EC.sub.50 determinations of necroptosis
inhibition in FADD-deficient Jurkat T cells treated with
TNF-.alpha. by compounds of the invention having formula (I-a). The
standard deviation is <10%.
TABLE-US-00004 TABLE 2 (I-a) ##STR00093## Compound R.sup.1 R.sup.2
R.sup.3 EC.sub.50 (.mu.M) 25 Me H 2-Cl-6-F-Ph 1.0 26 Me Me
2-Cl-6-F-Ph 11 27 Me H 2,6-di-F-Ph 3.5 28 Me H 2-Me-Ph 27 29 Me H
2-OMe-Ph >100 30 n-Pr H 2-Cl-6-F-Ph 4.1 31 i-Pr H 2-Cl-6-F-Ph
0.58 32 c-Pr H 2-Cl-6-F-Ph 0.50 33 c-Bu H 2-Cl-6-F-Ph 0.60 34
c-Pentyl H 2-Cl-6-F-Ph 1.9 35 c-Hex H 2-Cl-6-F-Ph 6.0 36 t-Bu H
2-Cl-6-F-Ph 18 37 Ph H 2-Cl-6-F-Ph >100 38 c-Pr H 2,6-di-Cl-Ph
6.0 39 c-Pr H 2,6-di-F-Ph 1.5 40 c-Pr H 2-F-Ph 1.5 41 c-Pr H
2-Cl-6-Me-Ph 10 42 c-Pr H 2-Cl-6-(OPh)-Ph >100 43 c-Pr H
2-Cl-6-CN-Ph >100 44 c-Pr H 2-F-6-CF.sub.3-Ph >100 45 c-Pr H
1-naphthyl >100 46 c-Pr H 2-Py 40 47 c-Pr H 3-F-2-Py 9.6 48 c-Pr
H 2-Cl-3,6-di-F-Ph 0.52 49 c-Pr H 3-Cl-2,6-di-F-Ph 0.18 50 c-Pr Me
2-Cl-6-F-Ph 16 c-Pr = cyclopropyl; c-Bu = cyclobutyl; c-Hex =
cyclohexyl; Py = pyridyl
[0192] The initial [1,2,3]thiadiazole necroptosis inhibitor
discovered during high throughput screening (HTS) was (25), with an
EC50=1 .mu.M (Table 2). Altering the amide NH, for example, through
simple methylation (26 vs 25 and 50 vs 32), resulted in significant
loss of activity. Introduction of branching into the alkyl group at
the 4-position of the [1,2,3]thiadiazole increased activity, with
i-Pr (31), c-Pr (32) and c-Bu (33) showing the best results.
However, introduction of a t-Bu (36) or phenyl (37) at this
position resulted in loss of activity. The 2-chloro-6-fluoro
substitution of the phenyl ring also appeared to be necessary for
potent activity. For example, compounds with a 2-methylphenyl (28)
or 2-methoxyphenyl (29) were less active. In addition, the
2,6-dichloro (38) or 2,6-difluoro (39) substituted derivatives were
also less active compared to the 2-chloro-6-fluoro substitution
(32). Consistent with these findings, removing one of the halogens
(40) or replacing one of the halogens with small (41) or large (42)
electron donating groups also resulted in decreased activity.
Replacing one of the halogens with other electron withdrawing
groups, such as cyano (43) or CF3 (44), did not restore activity.
Replacing the 2-chloro-6-fluorophenyl with a 1-naphthyl (45),
2-pyridyl (46) or substituted 2-pyridyl (47) was detrimental to
activity. However, addition of a halogen to the 3-position of the
2-chloro-6-fluorophenyl (49) gave an increase in necroptosis
inhibition activity with an EC50 value of 0.18 .mu.M.
[0193] Additional changes to the linker between the
[1,2,3]thiadiazole and the 2-chloro-6-fluorophenyl were examined.
Table 3 provides the EC.sub.50 determinations of necroptosis
inhibition in FADD-deficient Jurkat T cells treated with
TNF-.alpha. by compounds having Formula (I-b). The corresponding
secondary amine (13) and imide (16) derivatives of 32 were
inactive. Also, the benzylamide was necessary, with the homologous
phenethyl amide (51) and the truncated anilide (52) being
significantly less active. Compound 52 was prepared in low yield
(10%) by allowing 15 to react with 2,6-difluoroaniline in THF and
pyridine at room temperature. The reaction was unsuccessful with
2-chloro-6-fluoroaniline presumably due to steric hindrance.
Introduction of a methyl group (53) onto the benzylic position gave
a slight increase in activity. Quite surprisingly, when the two
enantiomers of 53 were examined, all of the necroptosis activity
resided in the (S)-enantiomer (55). However, increasing the steric
bulk of the benzylic substituent to n-Bu (56), phenyl (57) or
gem-dimethyl (58) resulted in loss of activity.
TABLE-US-00005 TABLE 3 (I-b) ##STR00094## Compound X Y R (R)/(S)
EC.sub.50 (.mu.M).sup.a 13 CH.sub.2 CH.sub.2 Cl -- >100 16
C.dbd.O C.dbd.O Cl -- >100 51 C.dbd.O CH.sub.2CH.sub.2 Cl -- 27
52 C.dbd.O -- F -- >100 53 C.dbd.O CH(Me) Cl (R)/(S) 0.40 54
C.dbd.O CH(Me) Cl (R) >100 55 C.dbd.O CH(Me) Cl (S) 0.28 56
C.dbd.O CH(n-Bu) Cl (R)/(S) >100 57 C.dbd.O CH(Ph) Cl (R)/(S)
>100 58 C.dbd.O C(Me).sub.2 Cl -- >100
[0194] Finally, the [1,2,3]thiadiazole was examined. Table 4 shows
the EC.sub.50 determinations of necroptosis inhibition in
FADD-deficient Jurkat T cells treated with TNF-.alpha. using
compounds having the Formula (I-f). Replacement with a variety of
thiazoles (59-61) or an oxazole (62) was detrimental to activity.
Likewise, the pyridazine (63), which attempted to replace the
sulfur of the [1,2,3]thiadiazole with a --CH.dbd.CH--, was also
inactive. However, moderate activity could be obtained with a
variety of thiophene derivatives (64-74), except for the ethoxy
derivative 75 and the sulfone derivative 76. In two case (66 and
74) the necroptosis activity approached that seen for the most
potent [1,2,3]thiadiazoles. However, replacement of the
[1,2,3]thiadiazole with a furan (77) was less effective.
TABLE-US-00006 TABLE 4 (I-f) ##STR00095## Com- EC.sub.50 pound
X.sub.3 X.sub.2 X.sub.1 R.sup.1 R.sup.7 (.mu.M).sup.a 59 S CH N Me
H 20 60 S CHMe N Me H >100 61 S CH-(4-ClPh) N Me H >100 62 O
CH N Me H >100 63 CH.dbd.CH N N Me H >100 64 S CH CH Me H 7.0
65 S CH CH Me Me 3.9 66 S CH CBr Me H 0.75 67 S CH CCN Me H 1.2 68
S CH CH c-Pr H 5.1 69 S CH CMe Cl H 3.9 70 S CH CH Cl H 9.6 71 S
CMe CH H H 3.9 72 S CH CH H H 9.4 73 S CH CMe H H 3.7 74 S CH
C(CH.sub.2).sub.4 H 0.48 75 S CH CH OEt H >100 76 S CH CR.sup.4
Me H >100 77 O CH CH Me H 13 Note: R.sup.4 in Compound (76) is
SO.sub.2(4-chlorophenyl)
Example 8
Evaluation of Pyrrole Compounds
[0195] Evaluation of compounds 78-99 (Table 5) for necroptosis
inhibitory activity was also performed. For EC.sub.50 value
determinations, cells were treated with 10 ng/mL of human
TNF-.alpha. in the presence of increasing concentration of test
compounds for 24 hours followed by ATP-based viability
assessment.
[0196] Microsome stability was determined in pooled mouse liver
microsomes. Test compound (3 .mu.M final concentration) along with
0.5 mg/mL microsome protein and 1 mM NADPH was incubated for 0, 5,
15, 30 and 60 minutes. Incubation of test compound and microsomes
in the absence of NADPH served as a negative control. The samples
were quenched with methanol and centrifuged for 20 minutes at 2500
rpm to precipitate proteins. Sample supernatants were analyzed
(N=3) by LC/MS. The In peak area ratio (compound peak area/internal
standard peak area) was plotted against time and the slope of the
line determined to give the elimination rate constant
[k=(-1)(slope)]. The half life (t.sub.1/2 in minutes), and the in
vitro intrinsic clearance (CL.sub.int in .mu.L/min/mg protein) were
calculated according to the following equations, where V=incubation
volume in .mu.L/mg protein:
t 1 / 2 = 0.693 k ; CL int = V ( 0.693 ) t 1 / 2 . ##EQU00001##
[0197] The results of the biological studies are shown in Table
5.
TABLE-US-00007 TABLE 5 (I-d) ##STR00096## Com- CL.sub.int pound
EC.sub.50 (.mu.M).sup.a t.sub.1/2 (minutes) (.mu.L/minutes/mg
protein) 78 0.74 15.4 89.8 .+-. 3.0 79 >20 -- -- 80 >20 -- --
81 4.9 -- -- 82 >20 -- -- 83 7.8 -- -- 84 0.90 17.2 80.6 .+-.
4.4 85 0.44 -- -- 86 >20 -- -- 87 >20 -- -- 88 >20 -- --
89 2.1 -- -- 90 0.52 -- -- 91 >20 -- -- 92 >20 -- -- 93 2.4
-- -- 94 0.34 42.3 32.8 .+-. 2.2 95 1.9 -- -- 96 1.4 -- -- 97 0.092
236 5.9 .+-. 2.5 98 >20 -- -- 99 >20 -- --
Example 9
Determination of "Universal" and Diverse Cell
Type/Stimulus-Specific Necroptosis Inhibitors
[0198] The compounds described herein may show universal activity
in a broad range of necroptosis cellular systems or activity may be
restricted to specific cell types/stimuli. The compounds described
herein are expected to offer advantages, for example, under
conditions where molecule specificity may be beneficial, such as
treating chronic conditions like neurodegenerative diseases.
[0199] Activity may be demonstrated using the procedures known in
the art (see, for example, Teng et al., Bioorg. Med. Chem. Lett.,
15: 5039 (2005) and Jagtap et al., J. Med. Chem. 50: 1886 (2007)).
We performed similar analyses with the [1,2,3]thiadiazole series.
Compound 55 showed the same activity profile as necrostatins 1 and
2 (see Scheme 7 for structures) in Jurkat or L929 cells using
TNF-.alpha. as the necroptosis stimuli (FIG. 1). In addition, 55 is
fully active in mouse adult lung fibroblasts stimulated to undergo
necroptosis with a combination of TNF-.alpha. and zVAD.fmk.
##STR00097##
[0200] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each independent publication or patent
application was specifically and individually indicated to be
incorporated by reference.
[0201] While the invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications and this application is intended
to cover any variations, uses, or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present disclosure that come
within known or customary practice within the art to which the
invention pertains and may be applied to the essential features
hereinbefore set forth, and follows in the scope of the claims.
[0202] Other embodiments are within the claims.
* * * * *