U.S. patent application number 17/527536 was filed with the patent office on 2022-03-10 for compounds for repressing cancer cell growth.
The applicant listed for this patent is Institute For Cancer Research d/b/a The Research Institute Of Fox Chase Cancer Center, Institute For Cancer Research d/b/a The Research Institute Of Fox Chase Cancer Center. Invention is credited to Wafik S. El-Deiry, Liz J. Hernandez Borrero, Shengliang Zhang.
Application Number | 20220073519 17/527536 |
Document ID | / |
Family ID | |
Filed Date | 2022-03-10 |
United States Patent
Application |
20220073519 |
Kind Code |
A1 |
El-Deiry; Wafik S. ; et
al. |
March 10, 2022 |
Compounds For Repressing Cancer Cell Growth
Abstract
The disclosure generally relates to compounds, compositions, and
methods for the treatment of cancer by restoring the P53 pathway
signaling to repress cancer cell growth.
Inventors: |
El-Deiry; Wafik S.;
(Philadelphia, PA) ; Zhang; Shengliang;
(Philadelphia, PA) ; Hernandez Borrero; Liz J.;
(Philadelphia, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Institute For Cancer Research d/b/a The Research Institute Of Fox
Chase Cancer Center |
Philadelphia |
PA |
US |
|
|
Appl. No.: |
17/527536 |
Filed: |
November 16, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16255204 |
Jan 23, 2019 |
11203598 |
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17527536 |
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62621221 |
Jan 24, 2018 |
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62711142 |
Jul 27, 2018 |
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International
Class: |
C07D 473/08 20060101
C07D473/08; A61P 35/00 20060101 A61P035/00; A61K 45/06 20060101
A61K045/06; A61K 31/522 20060101 A61K031/522 |
Goverment Interests
STATEMENT OF GOVERNMENT SUPPORT
[0001] This invention was made with government support under Grant
No. CN043302 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1-22. (canceled)
23. A compound of Formula I: ##STR00024## or a pharmaceutically
acceptable salt thereof, wherein: R.sub.1 is hydrogen or a
substituted or unsubstituted haloalkyl group; each R.sub.2,
R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is, independently,
hydrogen, halogen, hydroxyl, cyano, substituted or unsubstituted
alkyl, substituted or unsubstituted alkenyl, substituted or
unsubstituted alkoxy, substituted or unsubstituted aryl,
substituted or unsubstituted heteroaryl, substituted or
unsubstituted phosphate, substituted or unsubstituted
phosphoramidate, substituted or unsubstituted amine, substituted or
unsubstituted alkylamino, substituted or unsubstituted acylamino,
substituted or unsubstituted aminoalkoxy, or substituted or
unsubstituted alkylthio; or a compound of Formula II: ##STR00025##
or a pharmaceutically acceptable salt thereof, wherein: R.sub.1 is
hydrogen or a substituted or unsubstituted alkyl group; each
R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is,
independently, hydrogen, halogen, hydroxyl, substituted or
unsubstituted alkyl, substituted or unsubstituted alkoxy,
substituted or unsubstituted aryl, or substituted or unsubstituted
amine; X is sulfur, oxygen, or --NH; and n is an integer from 0 to
5; or a compound of Formula III: ##STR00026## or a pharmaceutically
acceptable salt thereof, wherein: R.sub.1 and R.sub.2 are,
independently, hydrogen or a substituted or unsubstituted alkyl;
R.sub.3 is a substituted or unsubstituted heteroaryl; and n is an
integer from 0 to 5.
24. A method of treating a cancer in a mammal comprising
administering to the mammal in need thereof a compound of Formula
I: ##STR00027## or a pharmaceutically acceptable salt thereof,
wherein: R.sub.1 is hydrogen or a substituted or unsubstituted
haloalkyl group; each R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6,
and R.sub.7 is, independently, cyano, substituted or unsubstituted
alkenyl, substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, substituted or unsubstituted phosphate,
substituted or unsubstituted phosphoramidate, substituted or
unsubstituted amine, substituted or unsubstituted alkylamino,
substituted or unsubstituted acylamino, substituted or
unsubstituted aminoalkoxy, or substituted or unsubstituted
alkylthio.
25. The method of claim 24, wherein the cancer is a tumor
suppressor protein p53 mutated associated cancer, tumor suppressor
protein Rb mutated associated cancer, tumor suppressor protein NF1
mutated associated cancer, tumor suppressor protein p16 mutated
associated cancer, tumor suppressor protein p27 mutated associated
cancer, or tumor suppressor protein VHL mutated associated
cancer.
26. The method of claim 24, wherein the human is also administered
radiation therapy, a chemotherapeutic agent, an immunotherapeutic
agent, a lysosome inhibitor, or a calpain inhibitor, or any
combination thereof.
27. A method of treating a cancer in a mammal comprising
administering to the mammal in need thereof a compound of Formula
II: ##STR00028## or a pharmaceutically acceptable salt thereof,
wherein: R.sub.1 is hydrogen or a substituted or unsubstituted
alkyl group; each R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and
R.sub.7 is, independently, hydrogen, halogen, hydroxyl, substituted
or unsubstituted alkyl, substituted or unsubstituted alkoxy,
substituted or unsubstituted aryl, or substituted or unsubstituted
amine; X is sulfur, oxygen, or --NH; and n is an integer from 0 to
5.
28. The method of claim 27, wherein R.sub.1 is hydrogen or an
unsubstituted alkyl.
29. The method of claim 27, wherein each R.sub.2, R.sub.3, R.sub.4,
R.sub.5, R.sub.6, and R.sub.7 is, independently, hydrogen, halogen,
hydroxyl, substituted or unsubstituted alkyl, substituted or
unsubstituted alkoxy, substituted or unsubstituted aryl, or
substituted or unsubstituted amine.
30. The method of claim 27, wherein X is sulfur or oxygen.
31. The method of claim 27, wherein n is an integer from 0 to
3.
32. The method of claim 27, wherein: R.sub.1 is hydrogen or an
unsubstituted alkyl; each R.sub.2, R.sub.3, R.sub.4, R.sub.5,
R.sub.6, and R.sub.7 is, independently, hydrogen, halogen,
hydroxyl, substituted or unsubstituted alkyl, substituted or
unsubstituted alkoxy, substituted or unsubstituted aryl, or
substituted or unsubstituted amine; X is sulfur or oxygen; and n is
an integer from 0 to 3.
33. The method of claim 27, wherein the cancer is a tumor
suppressor protein p53 mutated associated cancer, tumor suppressor
protein Rb mutated associated cancer, tumor suppressor protein NF1
mutated associated cancer, tumor suppressor protein p16 mutated
associated cancer, tumor suppressor protein p27 mutated associated
cancer, or tumor suppressor protein VHL mutated associated
cancer.
34. The method of claim 27, wherein the human is also administered
radiation therapy, a chemotherapeutic agent, an immunotherapeutic
agent, a lysosome inhibitor, or a calpain inhibitor, or any
combination thereof.
35. A method of treating a cancer in a mammal comprising
administering to the mammal in need thereof a compound of Formula
III: ##STR00029## or a pharmaceutically acceptable salt thereof,
wherein: R.sub.1 and R.sub.2 are, independently, hydrogen or a
substituted or unsubstituted alkyl; R.sub.3 is a substituted or
unsubstituted heteroaryl; and n is an integer from 0 to 5.
36. The method of claim 35, wherein R.sub.1 and R.sub.2 are,
independently, hydrogen or an unsubstituted alkyl.
37. The method of claim 35, wherein n is an integer from 0 to
3.
38. The method of claim 35, wherein R.sub.3 is an unsubstituted
heteroaryl.
39. The method of claim 35, wherein: R.sub.1 and R.sub.2 are,
independently, hydrogen or an unsubstituted alkyl; n is an integer
from 0 to 3; and R.sub.3 is an unsubstituted heteroaryl.
40. The method of claim 35, wherein the cancer is a tumor
suppressor protein p53 mutated associated cancer, tumor suppressor
protein Rb mutated associated cancer, tumor suppressor protein NF1
mutated associated cancer, tumor suppressor protein p16 mutated
associated cancer, tumor suppressor protein p27 mutated associated
cancer, or tumor suppressor protein VHL mutated associated
cancer.
41. The method of claim 35, wherein the human is also administered
radiation therapy, a chemotherapeutic agent, an immunotherapeutic
agent, a lysosome inhibitor, or a calpain inhibitor, or any
combination thereof.
Description
FIELD
[0002] The disclosure generally relates to compounds, compositions,
and methods for the treatment of cancer by restoring the P53
pathway signaling to repress cancer cell growth. The disclosure
provides novel P53 pathway restoring compounds, pharmaceutical
compositions containing such compounds, and methods for using these
compounds in the treatment of cancer.
BACKGROUND
[0003] Various publications, including patents, published
applications, technical articles and scholarly articles are cited
throughout the specification. Each of these cited publications is
incorporated by reference herein, in its entirety and for all
purposes.
[0004] The TP53 gene encodes the tumor suppressor protein p53,
known as "the guardian of the genome," which ensures the fidelity
of DNA replication and controls cell division, thereby preventing
the formation and abnormal growth of cancerous cells. p53 becomes
stimulated upon genotoxic and other cellular stress signals
including DNA damage, loss of cell adhesion, spindle damage,
oncogene activation, nutrient deprivation, ribonucleotide
depletion, and hypoxia. Ultimately, such stresses lead to
p53-mediated transcriptional activation of genes involved in DNA
repair, cell cycle arrest, cellular senescence, and apoptosis. One
of the most well studied outcomes of p53 has been apoptosis, owing
to p53's irreversible capacity to induce programmed cell death.
Among established p53 targets that participate in apoptosis are
NOXA, PUMA, DR5, and Bax.
[0005] TP53 is mutated in more than 50% of all human cancers, and
is one of the most explored cancer targets. TP53 mutation is a poor
prognostic marker in various types of cancer. Unlike other tumor
suppressors, missense mutations are the most common in TP53 and can
result in the expression of a stable mutated p53 protein. TP53
mutations can result in loss of function (LOF), a dominant-negative
phenotype, or gain of function (GOF) activity for the encoded
mutant protein. Studies have shown in vitro and in vivo that
introduction of certain types of p53 mutants in a p53-null
background results in new phenotypes where tumor cells are more
proliferative, invasive, resistant to therapy, or more
metastatic.
[0006] In addition to mutant p53 acting in a dominant-negative
fashion towards wild-type p53, mutant p53 has been shown to inhibit
p53 family proteins p73 and p63. Consequently, p73 and p63 become
incapable of exerting their tumor suppressive functions. p73 and
p63 are transcription factors that share significant structural
homology with p53. Similar to p53, p73 and p63 control the
expression of genes involved in cell cycle arrest and apoptosis. It
has been shown that p73 and p63 can functionally replace p53.
Unlike p53, however, they are very rarely mutated in cancer.
Therefore, restoration of the p53 pathway through its family
members represents an attractive therapeutic approach.
[0007] Despite numerous efforts to identify small molecule
compounds for mutant p53-targeted therapy, to date there is no
approved drug that restores a functional p53 pathway in cancer
cells with mutant p53. Given that TP53 is the most commonly mutated
tumor suppressor, it is an attractive therapeutic strategy to
identify such small molecules. With our current knowledge that p53
family members p73 and p63 can perform similar anti-tumor effects,
our group and others have identified small molecules that restore
the p53 pathway through the activation of p73. Using a
luciferase-based p53-reporter, our group has previously identified
several compounds that restore the p53 pathway including
prodigiosin and NSC59984. These compounds have been reported to
up-regulate p73 although the downstream mechanisms of action are
believed to be different, and other regulatory activities of the
molecules may be important. Furthermore, it is believed that mutant
p53 protein degradation is necessary for optimal p73-mediated p53
pathway restoration. These findings support the pursuit of
therapeutic strategies that target mutant p53 for degradation.
[0008] P53-targeted therapy is challenging because direct
functional restoration of p53 activity as a DNA-binding
transcription factor has been difficult to achieve using approaches
whose goal is to modify p53 protein structure. Investigating small
molecules that functionally restore the p53-signaling pathway
instead of requiring direct p53 protein binding has begun. It is
hypothesized that adequate p53 restoration in cancer cells carrying
mutated p53 may involve the removal or inactivation of mutant p53
protein and activation of p53 family members p73 and p63.
Accordingly, there is a need in the art for a compound or class of
compounds that targets and restores the p53 pathway in p53-null,
wild-type, or mutant p53-expressing cancer cells.
SUMMARY
[0009] The present disclosure provides compounds of Formula I:
##STR00001##
or a pharmaceutically acceptable salt thereof, wherein: R.sub.1 is
hydrogen or a substituted or unsubstituted haloalkyl group; and
each R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is,
independently, hydrogen, halogen, hydroxyl, cyano, substituted or
unsubstituted alkyl, substituted or unsubstituted alkenyl,
substituted or unsubstituted alkoxy, substituted or unsubstituted
aryl, substituted or unsubstituted heteroaryl, substituted or
unsubstituted phosphate, substituted or unsubstituted
phosphoramidate, substituted or unsubstituted amine, substituted or
unsubstituted alkylamino, substituted or unsubstituted acylamino,
substituted or unsubstituted aminoalkoxy, or substituted or
unsubstituted alkylthio.
[0010] The present disclosure also provides pharmaceutical
compositions comprising one or more of the compounds of Formula I
and a pharmaceutically acceptable carrier.
[0011] The present disclosure also provides methods of treating a
cancer in a mammal comprising administering to the mammal in need
thereof a compound of Formula I.
[0012] The present disclosure also provides methods of restoring
the tumor suppressor protein p53 signaling pathway within a tumor
cell of a mammal comprising administering to the mammal in need
thereof a compound of Formula I.
[0013] The present disclosure also provides compounds of Formula
Ia:
##STR00002##
or a pharmaceutically acceptable salt thereof, wherein: R.sub.1 is
hydrogen or a substituted or unsubstituted alkyl; each R.sub.2,
R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is, independently,
hydrogen, halogen, hydroxyl, cyano, substituted or unsubstituted
alkyl, substituted or unsubstituted alkenyl, substituted or
unsubstituted haloalkyl, substituted or unsubstituted alkoxy,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, substituted or unsubstituted phosphate, substituted or
unsubstituted phosphoramidate, substituted or unsubstituted amine,
substituted or unsubstituted alkylamino, substituted or
unsubstituted acylamino, substituted or unsubstituted aminoalkoxy,
or substituted or unsubstituted alkylthio; and n is an integer from
0 to 5. In some embodiments, the compound of Formula Ia is not
##STR00003##
[0014] The present disclosure also provides pharmaceutical
compositions comprising one or more of the compounds of Formula Ia
and a pharmaceutically acceptable carrier.
[0015] The present disclosure also provides methods of treating a
cancer in a mammal comprising administering to the mammal in need
thereof a compound of Formula Ia.
[0016] The present disclosure also provides methods of restoring
the tumor suppressor protein p53 signaling pathway within a tumor
cell of a mammal comprising administering to the mammal in need
thereof a compound of Formula Ia.
[0017] The present disclosure also provides compounds of Formula
II:
##STR00004##
or a pharmaceutically acceptable salt thereof, wherein: R.sub.1 is
hydrogen or a substituted or unsubstituted alkyl group; each
R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is,
independently, hydrogen, halogen, hydroxyl, substituted or
unsubstituted alkyl, substituted or unsubstituted alkoxy,
substituted or unsubstituted aryl, or substituted or unsubstituted
amine; X is sulfur, oxygen, or --NH; and n is an integer from 0 to
5.
[0018] The present disclosure also provides pharmaceutical
compositions comprising one or more of the compounds of Formula II
and a pharmaceutically acceptable carrier.
[0019] The present disclosure also provides methods of treating a
cancer in a mammal comprising administering to the mammal in need
thereof a compound of Formula II.
[0020] The present disclosure also provides methods of restoring
the tumor suppressor protein p53 signaling pathway within a tumor
cell of a mammal comprising administering to the mammal in need
thereof a compound of Formula II.
[0021] The present disclosure also provides compound of Formula
III:
##STR00005##
or a pharmaceutically acceptable salt thereof, wherein: R.sub.1 and
R.sub.2 are, independently, hydrogen or a substituted or
unsubstituted alkyl; R.sub.3 is a substituted or unsubstituted
heteroaryl; and n is an integer from 0 to 5.
[0022] The present disclosure also provides pharmaceutical
compositions comprising one or more of the compounds of Formula III
and a pharmaceutically acceptable carrier.
[0023] The present disclosure also provides methods of treating a
cancer in a mammal comprising administering to the mammal in need
thereof a compound of Formula III.
[0024] The present disclosure also provides methods of restoring
the tumor suppressor protein p53 signaling pathway within a tumor
cell of a mammal comprising administering to the mammal in need
thereof a compound of Formula III.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1 (Panels A-E) shows CB002 activation of
luciferase-based p53-reporter activity in three different human
colorectal cancer cell lines in a dose-dependent manner.
[0026] FIG. 2 shows CB002 has a favorable therapeutic index in
cancer cell lines.
[0027] FIG. 3 shows CB002 qualitatively decreases colony formation
in SW480, DLD-1, and DLD-1 stable p73 knockdown colorectal cancer
cell lines.
[0028] FIG. 4 shows CB002 treatment for 48 hours increases
apoptotic cells as indicated by the sub-G1 content in SW480 cancer
cells but not in normal WI38 cells.
[0029] FIG. 5 (Panels A-E) shows CB002 induces expression of p53
target genes independently of p73 and NOXA is required for
CB002-mediated apoptosis.
[0030] FIG. 6 (Panels A-C) shows CB002 does not reduce mutant p53
stability in SW480 and DLD-1 cells.
[0031] FIG. 7 (Panels A-D) shows CB002 treatment reduces the
stability of the R175H p53 mutant.
[0032] FIG. 8 (Panels A-F) shows CB002-mediated autophagy
contributes to apoptotic cell death in drug treated cells.
[0033] FIG. 9 (Panels A-F) shows screening experimental compounds
CB002 and Analog 11 for transcriptional activation by p53 in cancer
cells.
[0034] FIG. 10 (Panels A-C) shows the effect of CB002 and Analog 11
on p53 pathway signaling in cancer cells.
[0035] FIG. 11 shows p53 protein level in cancer cells upon CB002
and Analog 11 treatment.
[0036] FIG. 12 (Panels A-D) shows CB002 and Analog 11 induce cell
death in colorectal cancer cells.
[0037] FIG. 13 (Panels A-C) shows CB002 induces cell death in tumor
cells with no significant effect on normal cells.
[0038] FIG. 14 (Panels A-E) shows synergistic effects of CB002 and
CPT-11 or 5-FU in treated cancer cells.
[0039] FIG. 15 shows structural analogs activate p53 reporter
activity in SW480 cells in a dose dependent manner (6 hours).
[0040] FIG. 16 shows therapeutic indexes for structural analogs was
determined in SW480 cells.
[0041] FIG. 17 shows Analog 4 increased apoptotic cells as
indicated by the Sub-G.sub.1 content. Two-way ANOVA, ***p<0.05,
****p<0.0001.
[0042] FIG. 18 shows ganetespib treatment enhances CB002 mediated
mutant p53 degradation in RXF393 cells (16 hours).
[0043] FIG. 19 (Panels A and B) shows CB002 and Analog 11 synergize
with ganetespib (Panel A) and Irinotecan (Panel B), respectively.
SW480 cells were treated as indicated for 24 hours.
[0044] FIG. 20 (Panels A-C) shows Analog 4 restores the p53 pathway
in SW480 cells, resulting in PARP cleavage (Panel A); NOXA
expression is increased by CB002 and derivatives at 24 hours in
SW480 cells (Panel B); 8226 bortezomib resistant multiple myeloma
cells (Panel C).
[0045] FIG. 21 (Panels A-C) shows 24 hours treatment of bortezomib
in combination with structural Analog 4 (Panel A); Analog 11 (Panel
B); synergistic cell death by Analog 4 and bortezomib at 48 hours
as indicated by the Cl. Bortezomib therapeutic indexes for MM1S
cell line at 36 hours (Panel C).
[0046] FIG. 22 (Panels A-F) shows propidium iodide stating cell
cycle analysis of CB002, etoposide, and caffeine treatment.
[0047] FIG. 23 (Panels A and B) shows Noxa induction in SW80 and
DLD-1 cells treated with CB002, Analog 4, caffeine, pentoxifylline,
or theophylline.
DESCRIPTION OF EMBODIMENTS
[0048] Various terms relating to aspects of the disclosure are used
throughout the specification and claims. Such terms are to be given
their ordinary meaning in the art, unless otherwise indicated.
Other specifically defined terms are to be construed in a manner
consistent with the definition provided herein. Unless defined
otherwise, all technical and scientific terms have the same meaning
as is commonly understood by one of ordinary skill in the art to
which the disclosed embodiments belong.
[0049] As used herein, the terms "a" or "an" mean "at least one" or
"one or more" unless the context clearly indicates otherwise.
[0050] As used herein, the term "about" means that the recited
numerical value is approximate and small variations would not
significantly affect the practice of the disclosed embodiments.
Where a numerical value is used, unless indicated otherwise by the
context, "about" means the numerical value can vary by .+-.10% and
remain within the scope of the disclosed embodiments.
[0051] As used herein, the term "acylamino" means an amino group
substituted by an acyl group. Examples of acylamino groups include,
but are not limited to, --NHC(.dbd.O)H and --NHC(.dbd.O)CH.sub.3.
The phrase "lower acylamino" refers to an amino group substituted
by a lower acyl group (e.g., --R--C(.dbd.O)--H or
--R--C(.dbd.O)--C.sub.1-6alkyl). Examples of lower acylamino groups
include, but are not limited to, --NHC(.dbd.O)H,
--NHC(.dbd.O)CH.sub.3, --NHC(.dbd.O)CH.sub.2CH.sub.3,
--NHC(.dbd.O)(CH.sub.2).sub.2CH.sub.3,
--NHC(.dbd.O)(CH.sub.2).sub.3CH.sub.3,
--NHC(.dbd.O)(CH.sub.2).sub.4CH.sub.3, and
--NHC(.dbd.O)(CH.sub.2).sub.5CH.sub.3.
[0052] As used herein, the term "alkenyl" means a straight or
branched alkyl group having 2 to 20 carbon atoms and having one or
more double carbon-carbon bonds. In some embodiments, the alkenyl
group has from 2 to 10 carbon atoms, from 2 to 8 carbon atoms, from
2 to 6 carbon atoms, from 2 to 4 carbon atoms, from 3 to 10 carbon
atoms, from 3 to 8 carbon atoms, from 3 to 6 carbon atoms, or 3 or
4 carbon atoms. Examples of alkenyl groups include, but are not
limited to, ethenyl, 1-propenyl, 2-methyl-1-propenyl, 2-propenyl,
1-butenyl, 2-butenyl, and the like.
[0053] As used herein, the term "alkoxy" means a straight or
branched --O-alkyl group having 1 to 20 carbon atoms. In some
embodiments, the alkoxy group has from 1 to 10 carbon atoms, from 1
to 8 carbon atoms, from 1 to 6 carbon atoms, from 1 to 4 carbon
atoms, from 2 to 10 carbon atoms, from 2 to 8 carbon atoms, from 2
to 6 carbon atoms, or from 2 to 4 carbon atoms. Examples of alkoxy
groups include, but are not limited to, methoxy, ethoxy, n-propoxy,
isopropoxy, t-butoxy, and the like.
[0054] As used herein, the term "alkyl" means a saturated
hydrocarbon group which is straight-chained or branched. In some
embodiments, the alkyl group has from 1 to 20 carbon atoms, from 2
to 20 carbon atoms, from 1 to 10 carbon atoms, from 2 to 10 carbon
atoms, from 1 to 8 carbon atoms, from 2 to 8 carbon atoms, from 1
to 6 carbon atoms, from 2 to 6 carbon atoms, from 1 to 4 carbon
atoms, from 2 to 4 carbon atoms, from 1 to 3 carbon atoms, or 2 or
3 carbon atoms. Examples of alkyl groups include, but are not
limited to, methyl (Me), ethyl (Et), propyl (e.g., n-propyl and
isopropyl), butyl (e.g., n-butyl, t-butyl, isobutyl), pentyl (e.g.,
n-pentyl, isopentyl, neopentyl), hexyl, isohexyl, heptyl, octyl,
nonyl, 4,4-dimethylpentyl, 2,2,4-trimethylpentyl, decyl, undecyl,
dodecyl, 2-methyl-1-propyl, 2-methyl-2-propyl, 2-methyl-1-butyl,
3-methyl-1-butyl, 2-methyl-3-butyl, 2-methyl-1-pentyl,
2,2-dimethyl-1-propyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl,
2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl,
2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, and
the like.
[0055] As used herein, the term "alkylamino" means an amino group
substituted by an alkyl group. In some embodiments, the alkyl group
is a lower alkyl group having from 1 to 6 carbon atoms. Alkylamino
groups include, but are not limited to, --NHCH.sub.2CH.sub.3,
--NH(CH.sub.2).sub.2CH.sub.3, --NH(CH.sub.2).sub.3CH.sub.3,
--NH(CH.sub.2).sub.4CH.sub.3, and --NH(CH.sub.2).sub.5CH.sub.3, and
the like.
[0056] As used herein, the term "alkylene" or "alkylenyl" means a
divalent alkyl linking group. Example of alkylenes (or alkylenyls)
include, but are not limited to, methylene or methylenyl
(--CH.sub.2--), ethylene or ethylenyl (--CH.sub.2--CH.sub.2--), and
propylene or propylenyl (--CH.sub.2--CH.sub.2--CH.sub.2--).
[0057] As used herein, the term "alkylthio" means an --S-alkyl
group having from 1 to 6 carbon atoms. Alkylthio groups include,
but are not limited to, --SCH.sub.2CH.sub.3,
--S(CH.sub.2).sub.2CH.sub.3, --S(CH.sub.2).sub.3CH.sub.3,
--S(CH.sub.2).sub.4CH.sub.3, and --S(CH.sub.2).sub.5CH.sub.3, and
the like.
[0058] As used herein, the term "alkynyl" means a straight or
branched alkyl group having 2 to 20 carbon atoms and one or more
triple carbon-carbon bonds. In some embodiments, the alkynyl group
has from 2 to 10 carbon atoms, from 2 to 8 carbon atoms, from 2 to
6 carbon atoms, or from 2 to 4 carbon atoms. Examples of alkynyl
groups include, but are not limited to, acetylene, 1-propylene,
2-propylene, and the like.
[0059] As used herein, the term "amidino" means
--C(.dbd.NH)NH.sub.2.
[0060] As used herein, the term "amino" means --NH.sub.2.
[0061] As used herein, the term "aminoalkoxy" means an alkoxy group
substituted by an amino group. Examples of aminoalkoxy groups
include, but are not limited to, --OCH.sub.2NH.sub.2,
--OCH.sub.2CH.sub.2NH.sub.2, --O(CH.sub.2).sub.3NH.sub.2, and
--O(CH.sub.2).sub.4NH.sub.2, and the like.
[0062] As used herein, the term "aminoalkyl" means an alkyl group
substituted by an amino group. Examples of aminoalkyl groups
include, but are not limited to, --CH.sub.2NH.sub.2,
--CH.sub.2CH.sub.2NH.sub.2, --(CH.sub.2).sub.3NH.sub.2,
--(CH.sub.2).sub.4NH.sub.2, and the like.
[0063] As used herein, the term "animal" includes, but is not
limited to, mammals, humans and non-human vertebrates, such as
wild, domestic, and farm animals.
[0064] As used herein, the terms "antagonize" and "antagonizing"
mean reducing or completely eliminating one or more effects.
[0065] As used herein, the term "aryl" means a monocyclic,
bicyclic, or polycyclic (e.g., having 2, 3 or 4 fused rings)
aromatic hydrocarbon. In some embodiments, the aryl group has from
6 to 20 carbon atoms or from 6 to 10 carbon atoms. Examples of aryl
groups include, but are not limited to, phenyl, naphthyl,
anthracenyl, phenanthrenyl, indanyl, indenyl, and
tetrahydronaphthyl, and the like.
[0066] As used herein, the term "arylalkyl" means an alkyl group
substituted by an aryl. In some embodiments, the alkyl group is a
C.sub.1-6alkyl group.
[0067] As used herein, the term "arylamino" means an amino group
substituted by an aryl group. Examples of arylamino groups include,
but are not limited to, --NH(phenyl) and the like.
[0068] As used herein, the term "arylene" means an aryl linking
group, i.e., an aryl group that links one group to another group in
a molecule.
[0069] As used herein, the term "carrier" means a diluent,
adjuvant, or excipient with which a compound is administered in a
composition.
[0070] As used herein, the term, "compound" means all
stereoisomers, tautomers, isotopes, and polymorphs of the compounds
described herein.
[0071] As used herein, the terms "comprising" (and any form of
comprising, such as "comprise", "comprises", and "comprised"),
"having" (and any form of having, such as "have" and "has"),
"including" (and any form of including, such as "includes" and
"include"), or "containing" (and any form of containing, such as
"contains" and "contain"), are inclusive and open-ended and include
the options following the terms, and do not exclude additional,
unrecited elements or method steps.
[0072] As used herein, the term "contacting" means bringing
together two compounds, molecules, or entities in an in vitro
system or an in vivo system.
[0073] As used herein, the term "cyano" means --CN.
[0074] As used herein, the term "cycloalkyl" means non-aromatic
cyclic hydrocarbons including cyclized alkyl, alkenyl, and alkynyl
groups that have up to 20 ring-forming carbon atoms. Cycloalkyl
groups have from 3 to 15 ring-forming carbon atoms, from 3 to 10
ring-forming carbon atoms, from 3 to 8 ring-forming carbon atoms,
from 3 to 6 ring-forming carbon atoms, from 4 to 6 ring-forming
carbon atoms, from 3 to 5 ring-forming carbon atoms, or 5 or 6
ring-forming carbon atoms. Ring-forming carbon atoms of a
cycloalkyl group can be optionally substituted by oxo or sulfido.
Cycloalkyl groups include, but are not limited to, monocyclic or
polycyclic ring systems such as fused ring systems, bridged ring
systems, and spiro ring systems. In some embodiments, polycyclic
ring systems include 2, 3, or 4 fused rings. Examples of cycloalkyl
groups include, but are not limited to, cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl,
cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl,
norbornyl, norpinyl, norcamyl, adamantyl, and the like. Cycloalkyl
groups can also have one or more aromatic rings fused (having a
bond in common with) to the cycloalkyl ring such as, for example,
benzo or thienyl derivatives of pentane, pentene, hexane, and the
like (e.g., 2,3-dihydro-1H-indene-1-yl, or
1H-inden-2(3H)-one-1-yl).
[0075] As used herein, the term "halo" means halogen groups and
includes, but is not limited to, fluoro, chloro, bromo, and
iodo.
[0076] As used herein, the term "haloalkoxy" means an --O-haloalkyl
group. Examples of haloalkoxy groups include, but are not limited
to, --OCF.sub.3 and --OCCl.sub.3.
[0077] As used herein, the term "haloalkyl" means a C.sub.1-6alkyl
group having one or more halogen substituents. Examples of
haloalkyl groups include, but are not limited to, --CF.sub.3,
--C.sub.2F.sub.5, --CHF.sub.2, --CCl.sub.3, --CHCl.sub.2,
--C.sub.2Cl.sub.5, --CH.sub.2CF.sub.3, and the like.
[0078] As used herein, the term "heteroaryl" means an aromatic
heterocycle having up to 20 ring-forming atoms (e.g., C) and having
at least one heteroatom ring member (ring-forming atom) such as
sulfur, oxygen, or nitrogen. In some embodiments, the heteroaryl
group has at least one or more heteroatom ring-forming atoms, each
of which are, independently, sulfur, oxygen, or nitrogen. In some
embodiments, the heteroaryl group has from 3 to 20 ring-forming
atoms, from 3 to 10 ring-forming atoms, from 3 to 6 ring-forming
atoms, or from 3 to 5 ring-forming atoms. In some embodiments, the
heteroaryl group contains 2 to 14 carbon atoms, from 2 to 7 carbon
atoms, or 5 or 6 carbon atoms. In some embodiments, the heteroaryl
group has 1 to 4 heteroatoms, 1 to 3 heteroatoms, or 1 or 2
heteroatoms. Heteroaryl groups include monocyclic and polycyclic
(e.g., having 2, 3 or 4 fused rings) systems. Examples of
heteroaryl groups include, but are not limited to, pyridyl,
pyrimidinyl, pyrazinyl, pyridazinyl, pyridinyl (including
2-aminopyridine), triazinyl, furyl, quinolyl, isoquinolyl, thienyl,
imidazolyl, thiazolyl, indolyl (such as indol-3-yl), pyrryl,
oxazolyl, benzofuryl, benzothienyl, pyrazolyl, benzthiazolyl,
isoxazolyl, triazolyl (including 1,2,4-triazole, 1,2,3-triazole,
and 5-amino-1,2,4-triazole), tetrazolyl, indazolyl, isothiazolyl,
1,2,4-thiadiazolyl, benzothienyl, purinyl, carbazolyl, isoxazolyl,
benzimidazolyl, indolinyl, pyranyl, pyrazolyl, triazolyl,
oxadiazolyl (including 1,2,3-oxadiazole, 1,2,4-oxadiazole,
1,2,5-oxadiazole, 3-amino-1,2,4-oxadiazole, 1,3,4-oxadiazole),
thianthrenyl, indolizinyl, isoindolyl, isobenzofuranyl, pyrrolyl,
benzoxazolyl, xanthenyl, 2H-pyrrolyl, 3H-indolyl, 4H-quinolizinyl,
phthalazinyl, acridinyl, naphthyridinyl, quinazolinyl,
phenanthridinyl, perimidinyl, phenanthrolinyl, phenazinyl,
isothiazolyl, phenothiazinyl, isoxazolyl, furazanyl, phenoxazinyl
groups, and the like.
[0079] As used herein, the term "heteroarylalkyl" means a
C.sub.1-6alkyl group substituted by a heteroaryl group.
[0080] As used herein, the term "heteroarylamino" means an amino
group substituted by a heteroaryl group.
[0081] As used herein, the term "heteroarylene" means a heteroaryl
linking group, i.e., a heteroaryl group that links one group to
another group in a molecule.
[0082] As used herein, the term "heterocycle" or "heterocyclic
ring" means a 5- to 7-membered monocyclic or 7- to 10-membered
bicyclic ring system, any ring of which may be saturated or
unsaturated, and which ring consists of carbon atoms and from one
to three heteroatoms chosen from N, O and S, and wherein the N and
S heteroatoms may optionally be oxidized, and the N heteroatom may
optionally be quaternized, and including any bicyclic group in
which any of the above-defined heterocyclic rings is fused to a
benzene ring. Heterocycles include rings containing one oxygen or
sulfur, one to three nitrogen atoms, or one oxygen or sulfur
combined with one or two nitrogen atoms. The heterocyclic ring may
be attached at any heteroatom or carbon atom which results in the
creation of a stable structure. Examples of heterocyclic groups
include, but are not limited to, piperidinyl, piperazinyl,
2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolodinyl,
2-oxoazepinyl, azepinyl, pyrrolyl, 4-piperidonyl, pyrrolidinyl,
pyrazolyl, pyrazolidinyl, imidazolyl, imidazolinyl, pyridyl,
imidazolidinyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl,
oxazolidinyl, isoxazolyl, isoxazolidinyl, morpholinyl, thiazolyl,
thiazolidinyl, isothiazolyl, quinuclidinyl, isothiazolidinyl,
indolyl, quinolinyl, isoquinolinyl, benzimidazolyl, thiadiazoyl,
benzopyranyl, benzothiazolyl, benzoxazolyl, furyl, tetrahydrofuryl,
tetrahydropyranyl, thienyl, benzothienyl, thiamorpholinyl,
thiamorpholinyl sulfoxide, thiamorpholinyl sulfone, oxadiazolyl,
and the like.
[0083] As used herein, the term "heterocycloalkyl" means
non-aromatic heterocycles having up to 20 ring-forming atoms
including cyclized alkyl, alkenyl, and alkynyl groups, where one or
more of the ring-forming carbon atoms is replaced by a heteroatom,
such as an O, N, or S atom. Hetercycloalkyl groups can be
monocyclic or polycyclic (e.g., fused, bridged, or spiro systems).
In some embodiments, the heterocycloalkyl group has from 1 to 20
carbon atoms or from 3 to 20 carbon atoms. In some embodiments, the
heterocycloalkyl group contains 3 to 14 ring-forming atoms, 3 to 7
ring-forming atoms, or 5 or 6 ring-forming atoms. In some
embodiments, the heterocycloalkyl group has 1 to 4 heteroatoms, 1
to 3 heteroatoms, or 1 or 2 heteroatoms. In some embodiments, the
heterocycloalkyl group has 0 to 3 double bonds. In some
embodiments, the heterocycloalkyl group has 0 to 2 triple bonds.
Examples of heterocycloalkyl groups include, but are not limited
to, morpholino, piperazinyl, thiomorpholino, tetrahydrofuranyl,
tetrahydrothienyl, 2,3-dihydrobenzofuryl, piperidinyl,
1,3-benzodioxole, benzo-1,4-dioxane, pyrrolidinyl, isoxazolidinyl,
oxazolidinyl, isothiazolidinyl, pyrazolidinyl, thiazolidinyl,
imidazolidinyl, pyrrolidin-2-one-3-yl, and the like. In addition,
ring-forming carbon atoms and heteroatoms of a heterocycloalkyl
group can be optionally substituted by oxo or sulfido. For example,
a ring-forming S atom can be substituted by 1 or 2 oxo (form a S(O)
or S(O).sub.2). For another example, a ring-forming C atom can be
substituted by oxo (form carbonyl). Heterocycloalkyl groups can
also have one or more aromatic rings fused (having a bond in common
with) to the nonaromatic heterocyclic ring including, but not
limited to, pyridinyl, thiophenyl, phthalimidyl, naphthalimidyl,
and benzo derivatives of heterocycles such as, for example,
indolene, isoindolene,
5,6-dihydrothieno[2,3-c]pyridin-7(4H)-one-5-yl,
isoindolin-1-one-3-yl,
4,5,6,7-tetrahydrothieno[2,3-c]pyridine-5-yl, and
3,4-dihydroisoquinolin-1(2H)-one-3yl groups. Ring-forming carbon
atoms and heteroatoms of the heterocycloalkyl group can be
optionally substituted by oxo or sulfido.
[0084] As used herein, the term "hydroxy" or "hydroxyl" means an
--OH group.
[0085] As used herein, the term "hydroxyalkyl" or "hydroxylalkyl"
means an alkyl group substituted by a hydroxyl group. Examples of
hydroxylalkyl groups include, but are not limited to, --CH.sub.2OH
and --CH.sub.2CH.sub.2OH.
[0086] As used herein, the terms "individual," "subject," and
"patient," used interchangeably, mean any animal described
herein.
[0087] As used herein, the phrase "in need thereof" means that the
"individual," "subject," or "patient" has been identified as having
a need for the particular method, prevention, or treatment. In some
embodiments, the identification can be by any means of diagnosis.
In any of the methods, preventions, and treatments described
herein, the "individual," "subject," or "patient" can be in need
thereof. In some embodiments, the "individual," "subject," or
"patient" is in an environment or will be traveling to an
environment, or has traveled to an environment in which a
particular disease, disorder, or condition is prevelant.
[0088] As used herein, the term "integer" means a numerical value
that is a whole number. For example, an "integer from 1 to 5" means
1, 2, 3, 4, or 5.
[0089] As used herein, the term "isolated" means that the
compounds, or pharmaceutically acceptable salts thereof, described
herein are separated from other components of either: a) a natural
source, such as a plant or cell, such as a bacterial culture, or b)
a synthetic organic chemical reaction mixture, such as by
conventional techniques.
[0090] As used herein, the term "mammal" means a rodent (i.e., a
mouse, a rat, or a guinea pig), a monkey, a sheep, a cat, a dog, a
cow, a horse, a pig, or a human. In some embodiments, the mammal is
a human.
[0091] As used herein, the term "nitro" means --NO.sub.2.
[0092] As used herein, the term "n-membered", where n is an
integer, typically describes the number of ring-forming atoms in a
moiety, where the number of ring-forming atoms is n. For example,
pyridine is an example of a 6-membered heteroaryl ring and
thiophene is an example of a 5-membered heteroaryl ring.
[0093] As used used herein, the phrase "optionally substituted"
means that a substitution is optional and, therefore, includes both
unsubstituted and substituted atoms and moieties. A "substituted"
atom or moiety indicates that any hydrogen atom on the designated
compound or moiety can be replaced with a selection from the
indicated substituent groups, provided that the normal valency of
the designated compound or moiety is not exceeded, and that the
substitution results in a stable compound. For example, if a methyl
group is optionally substituted, then 1, 2, or 3 hydrogen atoms on
the carbon atom within the methyl group can be replaced with 1, 2,
or 3 of the recited substituent groups.
[0094] As used herein, the phrase "pharmaceutically acceptable"
means that the compounds, materials, compositions, and/or dosage
forms are within the scope of sound medical judgment and are
suitable for use in contact with tissues of humans and other
animals. In some embodiments, "pharmaceutically acceptable" means
approved by a regulatory agency of the Federal government or a
state government or listed in the U.S. Pharmacopeia or other
generally recognized pharmacopeia for use in animals, and more
particularly in humans. In some embodiments, the pharmaceutically
acceptable compounds, materials, compositions, and/or dosage forms
result in no persistent detrimental effect on the subject, or on
the general health of the subject being treated. However, it will
be recognized that transient effects, such as minor irritation or a
"stinging" sensation, are common with administration of medicament
and the existence of such transient effects is not inconsistent
with the composition, formulation, or ingredient (e.g., excipient)
in question.
[0095] As used herein, the phrase "pharmaceutically acceptable
salt(s)," includes, but is not limited to, salts of acidic or basic
groups. Compounds that are basic in nature are capable of forming a
wide variety of salts with various inorganic and organic acids.
Acids that may be used to prepare pharmaceutically acceptable acid
addition salts of such basic compounds are those that form
non-toxic acid addition salts, i.e., salts containing
pharmacologically acceptable anions including, but not limited to,
sulfuric, thiosulfuric, citric, maleic, acetic, oxalic,
hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate,
bisulfate, bisulfite, phosphate, acid phosphate, isonicotinate,
borate, acetate, lactate, salicylate, citrate, acid citrate,
tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate,
succinate, maleate, gentisinate, fumarate, gluconate, glucaronate,
saccharate, formate, benzoate, glutamate, methanesulfonate,
ethanesulfonate, benzenesulfonate, p-toluenesulfonate, bicarbonate,
malonate, mesylate, esylate, napsydisylate, tosylate, besylate,
orthophoshate, trifluoroacetate, and pamoate (i.e.,
1,1'-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Compounds that
include an amino moiety may form pharmaceutically acceptable salts
with various amino acids, in addition to the acids mentioned above.
Compounds that are acidic in nature are capable of forming base
salts with various pharmacologically acceptable cations. Examples
of such salts include, but are not limited to, alkali metal or
alkaline earth metal salts and, particularly, calcium, magnesium,
ammonium, sodium, lithium, zinc, potassium, and iron salts. Salts
also includes quaternary ammonium salts of the compounds described
herein, where the compounds have one or more tertiary amine
moiety.
[0096] As used herein, the term "phenyl" means --C.sub.6H.sub.5. A
phenyl group can be unsubstituted or substituted with one, two, or
three suitable substituents.
[0097] As used herein, the terms "prevention" or "preventing" mean
a reduction of the risk of acquiring a particular disease,
condition, or disorder.
[0098] As used herein, the term "prodrug" means a derivative of a
known direct acting drug, which derivative may have enhanced
delivery characteristics and therapeutic value as compared to the
active drug, and is transformed into the active drug by an
enzymatic or chemical process.
[0099] As used herein, the term "purified" means that when
isolated, the isolate contains at least 90%, at least 95%, at least
98%, at least 99%, or 100% of a compound described herein by weight
of the isolate.
[0100] As used herein, the phrase "quaternary ammonium salts" means
derivatives of the disclosed compounds with one or more tertiary
amine moieties wherein at least one of the tertiary amine moieties
in the parent compound is modified by converting the tertiary amine
moiety to a quaternary ammonium cation via alkylation (and the
cations are balanced by anions such as Cl.sup.-, CH.sub.3COO.sup.-,
and CF.sub.3COO.sup.-), for example methylation or ethylation.
[0101] As used herein, the phrase "solubilizing agent" means agents
that result in formation of a micellar solution or a true solution
of the drug.
[0102] As used herein, the term "solution/suspension" means a
liquid composition wherein a first portion of the active agent is
present in solution and a second portion of the active agent is
present in particulate form, in suspension in a liquid matrix.
[0103] As used herein, the phrase "substantially isolated" means a
compound that is at least partially or substantially separated from
the environment in which it is formed or detected.
[0104] As used herein, the phrase "suitable substituent" or
"substituent" means a group that does not nullify the synthetic or
pharmaceutical utility of the compounds described herein or the
intermediates useful for preparing them. Examples of suitable
substituents include, but are not limited to: C.sub.1-C.sub.6alkyl,
C.sub.1-C.sub.6alkenyl, C.sub.1-C.sub.6alkynyl,
C.sub.5-C.sub.6aryl, C.sub.1-C.sub.6alkoxy,
C.sub.3-C.sub.5heteroaryl, C.sub.3-C.sub.6cycloalkyl,
C.sub.5-C.sub.6aryloxy, --CN, --OH, oxo, halo, haloalkyl,
--NO.sub.2, --CO.sub.2H, --NH.sub.2, --NH(C.sub.1-C.sub.8alkyl),
--N(C.sub.1-C.sub.8alkyl).sub.2, --NH(C.sub.6aryl),
--N(C.sub.5-C.sub.6aryl).sub.2, --CHO, --CO(C.sub.1-C.sub.6alkyl),
--CO((C.sub.5-C.sub.6)aryl), --CO.sub.2((C.sub.1-C.sub.6)alkyl),
and --CO.sub.2((C.sub.5-C.sub.6)aryl). One of skill in art can
readily choose a suitable substituent based on the stability and
pharmacological and synthetic activity of the compounds described
herein.
[0105] As used herein, the phrase "therapeutically effective
amount" means the amount of active compound or pharmaceutical agent
that elicits the biological or medicinal response that is being
sought in a tissue, system, animal, individual or human by a
researcher, veterinarian, medical doctor, or other clinician. The
therapeutic effect is dependent upon the disorder being treated or
the biological effect desired. As such, the therapeutic effect can
be a decrease in the severity of symptoms associated with the
disorder and/or inhibition (partial or complete) of progression of
the disorder, or improved treatment, healing, prevention or
elimination of a disorder, or side-effects. The amount needed to
elicit the therapeutic response can be based on, for example, the
age, health, size, and sex of the subject. Optimal amounts can also
be determined based on monitoring of the subject's response to
treatment.
[0106] As used herein, the terms "treat," "treated," or "treating"
mean both therapeutic treatment and prophylactic or preventative
measures wherein the object is to prevent or slow down (lessen) an
undesired physiological condition, disorder or disease, or obtain
beneficial or desired clinical results. For purposes herein,
beneficial or desired clinical results include, but are not limited
to, alleviation of symptoms; diminishment of extent of condition,
disorder or disease; stabilized (i.e., not worsening) state of
condition, disorder or disease; delay in onset or slowing of
condition, disorder or disease progression; amelioration of the
condition, disorder or disease state or remission (whether partial
or total), whether detectable or undetectable; an amelioration of
at least one measurable physical parameter, not necessarily
discernible by the patient; or enhancement or improvement of
condition, disorder or disease. Treatment includes eliciting a
clinically significant response, optionally without excessive
levels of side effects. Treatment also includes prolonging survival
as compared to expected survival if not receiving treatment.
[0107] At various places herein, substituents of compounds may be
disclosed in groups or in ranges. It is specifically intended that
the disclosure include each and every individual subcombination of
the members of such groups and ranges. For example, the term
"C.sub.1-6alkyl" is specifically intended to individually disclose
methyl, ethyl, propyl, C.sub.4alkyl, C.sub.5alkyl, and
C.sub.6alkyl.
[0108] For compounds in which a variable appears more than once,
each variable can be a different moiety chosen from the Markush
group providing options for the variable. For example, where a
structure is described having two R groups that are simultaneously
present on the same compound, the two R groups can represent
different moieties chosen from the Markush group defined for R. In
another example, when an optionally multiple substituent "R" is
designated in the form, for example,
##STR00006##
then it should be understood that substituent "R" can occur "x"
number of times on the ring at any position(s), and "R" can be a
different moiety at each occurrence. Further, in the above example,
where the variable "Y" normally would include one or more
hydrogens, such as when "Y" is CH.sub.2, NH, etc., any H can be
replaced with a substituent.
[0109] It should be appreciated that particular features of the
disclosure, which are, for clarity, described in the context of
separate embodiments, can also be provided in combination in a
single embodiment. Conversely, various features of the disclosure
which are, for brevity, described in the context of a single
embodiment, can also be provided separately or in any suitable
subcombination.
[0110] It should be understood that stereoisomers (including
diastereomers and enantiomers) of the compounds described herein,
as well as mixtures thereof, are within the scope of the present
disclosure. By way of non-limiting example, the mixture may be a
racemate or the mixture may comprise unequal proportions of one
particular stereoisomer over the other. Additionally, the compounds
can be provided as a substantially pure stereoisomers.
Diastereomers include, for example, cis-trans isomers, E-Z isomers,
conformers, and rotamers. Methods of preparation of stereoisomers
are known in the art, such as by resolution of racemic mixtures or
by stereoselective synthesis. Many geometric isomers of olefins,
C.dbd.N double bonds, and the like can also be present in the
compounds described herein, and all such stable isomers are
contemplated in the present disclosure. Cis and trans geometric
isomers of the compounds are also included within the scope of the
disclosure and can be isolated as a mixture of isomers or as
separated isomeric forms. Where a compound capable of
stereoisomerism or geometric isomerism is designated in its
structure or name without reference to specific R/S or cis/trans
configurations, it is intended that all such isomers are
contemplated.
[0111] Resolution of racemic mixtures of compounds can be carried
out by any of numerous methods known in the art, including, for
example, fractional recrystallizaion using a chiral resolving acid
which is an optically active, salt-forming organic acid. Suitable
resolving agents for fractional recrystallization methods include,
but are not limited to, optically active acids, such as the D and L
forms of tartaric acid, diacetyltartaric acid, dibenzoyltartaric
acid, mandelic acid, malic acid, lactic acid, and the various
optically active camphorsulfonic acids such as
.beta.-camphorsulfonic acid. Other resolving agents suitable for
fractional crystallization methods include, but are not limited to,
stereoisomerically pure forms of .alpha.-methylbenzylamine (e.g., S
and R forms, or diastereomerically pure forms), 2-phenylglycinol,
norephedrine, ephedrine, N-methylephedrine, cyclohexylethylamine,
1,2-diaminocyclohexane, and the like. Resolution of racemic
mixtures can also be carried out by elution on a column packed with
an optically active resolving agent (e.g.,
dinitrobenzoylphenylglycine). Suitable elution solvent compositions
can be determined by one skilled in the art.
[0112] Appropriate compounds described herein may also include
tautomeric forms. Tautomeric forms result from the swapping of a
single bond with an adjacent double bond together with the
concomitant migration of a proton. Tautomeric forms include
prototropic tautomers which are isomeric protonation states having
the same empirical formula and total charge. Examples of
prototropic tautomers include, but are not limited to, ketone-enol
pairs, amide-imidic acid pairs, lactam-lactim pairs, amide-imidic
acid pairs, enamine-imine pairs, and annular forms where a proton
can occupy two or more positions of a heterocyclic system
including, but not limited to, 1H- and 3H-imidazole, 1H-, 2H- and
4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole.
Tautomeric forms can be in equilibrium or sterically locked into
one form by appropriate substitution.
[0113] The compounds described herein also include hydrates and
solvates, as well as anhydrous and non-solvated forms.
[0114] The compounds described herein can also include all isotopes
of atoms occurring in the intermediates or final compounds.
Isotopes include those atoms having the same atomic number but
different mass numbers. For example, isotopes of hydrogen include
tritium and deuterium. Carbon (.sup.12C) can be replaced at any
position with .sup.13C or .sup.14C. Nitrogen (.sup.14N) can be
replaced with .sup.15N. Oxygen (.sup.16O) can be replaced at any
position with .sup.17O or .sup.18O. Sulfur (.sup.32S) can be
replaced with .sup.33S, .sup.34S or .sup.36S. Chlorine (.sup.35Cl)
can be replaced with .sup.37Cl. Bromine (.sup.79Br) can be replaced
with .sup.81Br.
[0115] In some embodiments, the compounds, or salts thereof, are
substantially isolated. Partial separation can include, for
example, a composition enriched in any one or more of the compounds
described herein. Substantial separation can include compositions
containing at least about 50%, at least about 60%, at least about
70%, at least about 80%, at least about 90%, at least about 95%, at
least about 97%, or at least about 99% by weight of any one or more
of the compounds described herein, or salt thereof. Methods for
isolating compounds and their salts are routine in the art.
[0116] The compounds described herein also include derivatives
referred to as prodrugs, which can be prepared by modifying
functional groups present in the compounds in such a way that the
modifications are cleaved, either in routine manipulation or in
vivo, to the parent compounds. Examples of prodrugs include
compounds as described herein that contain one or more molecular
moieties appended to a hydroxyl, amino, sulfhydryl, or carboxyl
group of the compound, and that when administered to a patient,
cleaves in vivo to form the free hydroxyl, amino, sulfhydryl, or
carboxyl group, respectively. Examples of prodrugs include, but are
not limited to, acetate, formate and benzoate derivatives of
alcohol and amine functional groups in the compounds described
herein. Preparation and use of prodrugs is discussed in T. Higuchi
et al., "Pro-drugs as Novel Delivery Systems," Vol. 14 of the
A.C.S. Symposium Series, and in Bioreversible Carriers in Drug
Design, ed. Edward B. Roche, American Pharmaceutical Association
and Pergamon Press, 1987, both of which are incorporated herein by
reference in their entireties.
[0117] Compounds containing an amine function can also form
N-oxides. A reference herein to a compound that contains an amine
function also includes the N-oxide. Where a compound contains
several amine functions, one or more than one nitrogen atom can be
oxidized to form an N-oxide. Examples of N-oxides include N-oxides
of a tertiary amine or a nitrogen atom of a nitrogen-containing
heterocycle. N-Oxides can be formed by treatment of the
corresponding amine with an oxidizing agent such as hydrogen
peroxide or a per-acid (e.g., a peroxycarboxylic acid) (see,
Advanced Organic Chemistry, by Jerry March, 4th Edition, Wiley
Interscience).
[0118] The present disclosure provides compounds of Formula I:
##STR00007##
or a pharmaceutically acceptable salt thereof, wherein:
[0119] R.sub.1 is hydrogen or a substituted or unsubstituted
haloalkyl group; and
[0120] each of R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and
R.sub.7 is, independently, hydrogen, halogen, hydroxyl, cyano,
substituted or unsubstituted alkyl, substituted or unsubstituted
alkenyl, substituted or unsubstituted alkoxy, substituted or
unsubstituted aryl, substituted or unsubstituted heteroaryl,
substituted or unsubstituted phosphate, substituted or
unsubstituted phosphoramidate, substituted or unsubstituted amine,
substituted or unsubstituted alkylamino, substituted or
unsubstituted acylamino, substituted or unsubstituted aminoalkoxy,
or substituted or unsubstituted alkylthio.
[0121] In some embodiments, each of R.sub.2, R.sub.3, R.sub.4,
R.sub.5, R.sub.6, and R.sub.7 is, independently, hydrogen, halogen,
hydroxyl, substituted or unsubstituted alkyl, or substituted or
unsubstituted alkoxy. In some embodiments, each of R.sub.2,
R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is, independently,
hydrogen, halogen, or substituted or unsubstituted alkyl. In some
embodiments, each of R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6,
and R.sub.7 is hydrogen. In some embodiments, each of R.sub.2,
R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is not
hydrogen.
[0122] In some embodiments, R.sub.1 is --CF.sub.3,
--C.sub.2F.sub.5, --CHF.sub.2, --CCl.sub.3, --CHCl.sub.2,
--C.sub.2Cl.sub.5, or --CH.sub.2CF.sub.3.
[0123] In some embodiments, R.sub.1 is a substituted or
unsubstituted haloalkyl group; and each of R.sub.2, R.sub.3,
R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is independently, hydrogen,
halogen, hydroxyl, substituted or unsubstituted alkyl, or
substituted or unsubstituted alkoxy.
[0124] In some embodiments, R.sub.1 is --CF.sub.3,
--C.sub.2F.sub.5, --CHF.sub.2, --CCl.sub.3, --CHCl.sub.2,
--C.sub.2Cl.sub.5, or --CH.sub.2CF.sub.3; and each of R.sub.2,
R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is independently,
hydrogen, halogen, or substituted or unsubstituted alkyl.
[0125] In some embodiments, R.sub.1 is --CF.sub.3; and each of
R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is
hydrogen.
[0126] In some embodiments, R.sub.1 is --CF.sub.3; and each of
R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is not
hydrogen.
[0127] The present disclosure also provides compounds of Formula
Ia:
##STR00008##
or a pharmaceutically acceptable salt thereof, wherein:
[0128] R.sub.1 is hydrogen or a substituted or unsubstituted
alkyl;
[0129] each R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and
R.sub.7 is, independently, hydrogen, halogen, hydroxyl, cyano,
substituted or unsubstituted alkyl, substituted or unsubstituted
alkenyl, substituted or unsubstituted haloalkyl, substituted or
unsubstituted alkoxy, substituted or unsubstituted aryl,
substituted or unsubstituted heteroaryl, substituted or
unsubstituted phosphate, substituted or unsubstituted
phosphoramidate, substituted or unsubstituted amine, substituted or
unsubstituted alkylamino, substituted or unsubstituted acylamino,
substituted or unsubstituted aminoalkoxy, or substituted or
unsubstituted alkylthio; and
[0130] n is an integer from 0 to 5.
[0131] In some embodiments, the compound of Formula Ia is not
##STR00009##
[0132] In some embodiments, R.sub.1 is hydrogen, unsubstituted
alkyl, --CF.sub.3, --C.sub.2F.sub.5, --CHF.sub.2, --CCl.sub.3,
--CHCl.sub.2, --C.sub.2Cl.sub.5, or --CH.sub.2CF.sub.3. In some
embodiments, R.sub.1 is hydrogen or unsubstituted alkyl. In some
embodiments, R.sub.1 is hydrogen or methyl.
[0133] In some embodiments, each R.sub.2, R.sub.3, R.sub.4,
R.sub.5, R.sub.6, and R.sub.7 is, independently, hydrogen, halogen,
hydroxyl, cyano, substituted or unsubstituted alkyl, substituted or
unsubstituted haloalkyl, or substituted or unsubstituted alkoxy. In
some embodiments, each R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6,
and R.sub.7 is, independently, hydrogen, halogen, substituted or
unsubstituted alkyl, substituted or unsubstituted haloalkyl, or
substituted or unsubstituted alkoxy. In some embodiments, each
R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is,
independently, hydrogen, halogen, unsubstituted alkyl,
unsubstituted haloalkyl, or unsubstituted alkoxy. In some
embodiments, each R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and
R.sub.7 is, independently, hydrogen, F, Cl, methyl, --CF.sub.3, or
methoxy.
[0134] In some embodiments, n is an integer from 0 to 3. In some
embodiments, n is an integer from 0 to 2. In some embodiments, n is
0 or 1.
[0135] In some embodiments, R.sub.1 is hydrogen, unsubstituted
alkyl, --CF.sub.3, --C.sub.2F.sub.5, --CHF.sub.2, --CCl.sub.3,
--CHCl.sub.2, --C.sub.2Cl.sub.5, or --CH.sub.2CF.sub.3; n is an
integer from 0 to 3; and each R.sub.2, R.sub.3, R.sub.4, R.sub.5,
R.sub.6, and R.sub.7 is, independently, hydrogen, halogen,
hydroxyl, cyano, substituted or unsubstituted alkyl, substituted or
unsubstituted haloalkyl, or substituted or unsubstituted
alkoxy.
[0136] In some embodiments, R.sub.1 is hydrogen or unsubstituted
alkyl; n is an integer from 0 to 2; and each R.sub.2, R.sub.3,
R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is, independently, hydrogen,
halogen, substituted or unsubstituted alkyl, substituted or
unsubstituted haloalkyl, or substituted or unsubstituted
alkoxy.
[0137] In some embodiments, R.sub.1 is hydrogen or methyl; n is 0
or 1; and each R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and
R.sub.7 is, independently, hydrogen, halogen, unsubstituted alkyl,
unsubstituted haloalkyl, or unsubstituted alkoxy.
[0138] In some embodiments, R.sub.1 is hydrogen or methyl; n is 0
or 1; and each R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and
R.sub.7 is, independently, hydrogen, F, Cl, methyl, --CF.sub.3, or
methoxy.
[0139] In some embodiments, each R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is hydrogen; and n is 0.
[0140] In some embodiments, each R.sub.1, R.sub.2, R.sub.3,
R.sub.5, and R.sub.7 is hydrogen; each R.sub.4 and R.sub.6 is
methyl; and n is 0.
[0141] In some embodiments, each R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.6, and R.sub.7 is hydrogen; R.sub.5 is methoxy; and
n is 0.
[0142] In some embodiments, each R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.5, and R.sub.7 is hydrogen; R.sub.6 is --CF.sub.3;
and n is 0.
[0143] In some embodiments, each R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.6, and R.sub.7 is hydrogen; R.sub.5 is fluorine; and
n is 1.
[0144] In some embodiments, each R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.6, and R.sub.7 is hydrogen; R.sub.5 is methoxy; and
n is 1.
[0145] In some embodiments, each R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.6, and R.sub.7 is hydrogen; R.sub.5 is chlorine; and
n is 1.
[0146] In some embodiments, R.sub.1 is methyl; each R.sub.2,
R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is hydrogen; and n
is 0.
[0147] In some embodiments, R.sub.1 is methyl; each R.sub.2,
R.sub.3, R.sub.4, and R.sub.7 is hydrogen; R.sub.5 and R.sub.6 are
methyl; and n is 0.
[0148] The present disclosure also provides compounds of Formula
II:
##STR00010##
or a pharmaceutically acceptable salt thereof, wherein:
[0149] R.sub.1 is hydrogen or a substituted or unsubstituted alkyl
group;
[0150] each R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and
R.sub.7 is, independently, hydrogen, halogen, hydroxyl, substituted
or unsubstituted alkyl, substituted or unsubstituted alkoxy,
substituted or unsubstituted aryl, or substituted or unsubstituted
amine;
[0151] X is sulfur, oxygen, or --NH; and
[0152] n is an integer from 0 to 5.
[0153] In some embodiments, R.sub.1 is hydrogen or an unsubstituted
alkyl. In some embodiments, R.sub.1 is hydrogen or methyl. In some
embodiments, R.sub.1 is hydrogen.
[0154] In some embodiments, each R.sub.2, R.sub.3, R.sub.4,
R.sub.5, R.sub.6, and R.sub.7 is, independently, hydrogen, halogen,
hydroxyl, substituted or unsubstituted alkyl, substituted or
unsubstituted alkoxy, substituted or unsubstituted aryl, or
substituted or unsubstituted amine. In some embodiments, each
R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is,
independently, hydrogen, halogen, or substituted or unsubstituted
alkyl. In some embodiments, each R.sub.2, R.sub.3, R.sub.4,
R.sub.5, R.sub.6, and R.sub.7 is, independently, hydrogen, halogen,
or unsubstituted alkyl. In some embodiments, each R.sub.2, R.sub.3,
R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is, independently, hydrogen,
halogen, or methyl.
[0155] In some embodiments, X is sulfur or oxygen. In some
embodiments, X is sulfur.
[0156] In some embodiments, n is an integer from 0 to 3. In some
embodiments, n is an integer from 0 to 2. In some embodiments, n is
1 or 2.
[0157] In some embodiments, R.sub.1 is hydrogen or an unsubstituted
alkyl; each R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and
R.sub.7 is, independently, hydrogen, halogen, hydroxyl, substituted
or unsubstituted alkyl, substituted or unsubstituted alkoxy,
substituted or unsubstituted aryl, or substituted or unsubstituted
amine; X is sulfur or oxygen; and n is an integer from 0 to 3.
[0158] In some embodiments, R.sub.1 is hydrogen or methyl; each
R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is,
independently, hydrogen, halogen, or substituted or unsubstituted
alkyl; X is sulfur or oxygen; and n is an integer from 0 to 2.
[0159] In some embodiments, R.sub.1 is hydrogen; each R.sub.2,
R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is, independently,
hydrogen, halogen, or unsubstituted alkyl; X is sulfur; and n is 1
or 2.
[0160] In some embodiments, R.sub.1 is hydrogen; each R.sub.2,
R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is, independently,
hydrogen, halogen, or methyl; X is sulfur; and n is 1 or 2.
[0161] In some embodiments, each R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.6, and R.sub.7 is hydrogen; R.sub.5 is methyl; X is
sulfur; and n is 2.
[0162] In some embodiments, each R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.6, and R.sub.7 is hydrogen; R.sub.5 is chlorine; X
is sulfur; and n is 2.
[0163] The present disclosure also provides compounds of Formula
III:
##STR00011##
or a pharmaceutically acceptable salt thereof, wherein:
[0164] R.sub.1 and R.sub.2 are, independently, hydrogen or a
substituted or unsubstituted alkyl;
[0165] R.sub.3 is a substituted or unsubstituted heteroaryl;
and
[0166] n is an integer from 0 to 5.
[0167] In some embodiments, R.sub.1 and R.sub.2 are, independently,
hydrogen or an unsubstituted alkyl. In some embodiments, R.sub.1
and R.sub.2 are, independently, hydrogen or methyl. In some
embodiments, R.sub.1 and R.sub.2 are hydrogen.
[0168] In some embodiments, n is an integer from 0 to 3. In some
embodiments, n is an integer from 0 to 2. In some embodiments, n is
1.
[0169] In some embodiments, R.sub.3 is an unsubstituted heteroaryl.
In some embodiments, R.sub.3 is unsubstituted heteroaryl having
from 3 to 10 ring-forming atoms. In some embodiments, R.sub.3 is
unsubstituted heteroaryl having from 3 to 6 ring-forming atoms. In
some embodiments, R.sub.3 is unsubstituted heteroaryl having from 3
to 5 ring-forming atoms. In some embodiments, R.sub.3 is pyridyl,
pyrimidinyl, pyrazinyl, pyridazinyl, pyridinyl, triazinyl, furyl,
quinolyl, thienyl, isoquinolyl, imidazolyl, thiazolyl, indolyl,
pyrryl, oxazolyl, benzofuryl, benzothienyl, purinyl, pyrazolyl,
benzthiazolyl, isoxazolyl, triazolyl, tetrazolyl, indazolyl,
isothiazolyl, 1,2,4-thiadiazolyl, benzothienyl, carbazolyl,
isoxazolyl, benzimidazolyl, indolinyl, pyranyl, pyrazolyl,
triazolyl, oxadiazolyl, thianthrenyl, indolizinyl, isoindolyl,
isobenzofuranyl, pyrrolyl, benzoxazolyl, xanthenyl, 2H-pyrrolyl,
3H-indolyl, 4H-quinolizinyl, phthalazinyl, acridinyl,
naphthyridinyl, quinazolinyl, phenanthridinyl, perimidinyl,
phenanthrolinyl, phenazinyl, isothiazolyl, phenothiazinyl,
isoxazolyl, or furazanyl. In some embodiments, R.sub.3 is pyridyl,
pyrimidinyl, pyrazinyl, pyridazinyl, pyridinyl, triazinyl, furyl,
quinolyl, thienyl, isoquinolyl, imidazolyl, thiazolyl, indolyl,
pyrryl, oxazolyl, purinyl, benzimidazolyl, indolinyl, pyranyl,
pyrazolyl, triazolyl, oxadiazolyl, thianthrenyl, or pyrrolyl. In
some embodiments, R.sub.3 is 2-thienyl or 3-thienyl.
[0170] In some embodiments, R.sub.1 and R.sub.2 are, independently,
hydrogen or an unsubstituted alkyl; n is an integer from 0 to 3;
and R.sub.3 is an unsubstituted heteroaryl.
[0171] In some embodiments, R.sub.1 and R.sub.2 are, independently,
hydrogen or an unsubstituted alkyl; n is an integer from 0 to 3;
and R.sub.3 is unsubstituted heteroaryl having from 3 to 10
ring-forming atoms.
[0172] In some embodiments, R.sub.1 and R.sub.2 are, independently,
hydrogen or methyl; n is an integer from 0 to 2; and R.sub.3 is
unsubstituted heteroaryl having from 3 to 6 ring-forming atoms.
[0173] In some embodiments, R.sub.1 and R.sub.2 are, independently,
hydrogen or methyl; n is an integer from 0 to 2; and R.sub.3 is
unsubstituted heteroaryl having from 3 to 5 ring-forming atoms.
[0174] In some embodiments, R.sub.1 and R.sub.2 are hydrogen; n is
an integer from 0 to 2; and R.sub.3 is pyridyl, pyrimidinyl,
pyrazinyl, pyridazinyl, pyridinyl, triazinyl, furyl, quinolyl,
isoquinolyl, thienyl, imidazolyl, thiazolyl, indolyl, pyrryl,
oxazolyl, benzofuryl, benzothienyl, pyrazolyl, benzthiazolyl,
isoxazolyl, triazolyl, tetrazolyl, indazolyl, isothiazolyl,
1,2,4-thiadiazolyl, benzothienyl, purinyl, carbazolyl, isoxazolyl,
benzimidazolyl, indolinyl, pyranyl, pyrazolyl, triazolyl,
oxadiazolyl, thianthrenyl, indolizinyl, isoindolyl,
isobenzofuranyl, pyrrolyl, benzoxazolyl, xanthenyl, 2H-pyrrolyl,
3H-indolyl, 4H-quinolizinyl, phthalazinyl, acridinyl,
naphthyridinyl, quinazolinyl, phenanthridinyl, perimidinyl,
phenanthrolinyl, phenazinyl, isothiazolyl, phenothiazinyl,
isoxazolyl, or furazanyl.
[0175] In some embodiments, R.sub.1 and R.sub.2 are hydrogen; n is
1; and R.sub.3 is pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl,
pyridinyl, triazinyl, furyl, quinolyl, isoquinolyl, thienyl,
imidazolyl, thiazolyl, indolyl, pyrryl, oxazolyl, purinyl,
benzimidazolyl, indolinyl, pyranyl, pyrazolyl, triazolyl,
oxadiazolyl, thianthrenyl, or pyrrolyl.
[0176] In some embodiments, R.sub.1 and R.sub.2 are hydrogen; n is
1; and R.sub.3 is 2-thienyl or 3-thienyl.
[0177] In some embodiments, R.sub.1 and R.sub.2 are hydrogen;
R.sub.3 is -2-thienyl; and n is 1.
[0178] Compound formulas are as follows:
##STR00012## ##STR00013##
[0179] In some embodiments, the compound is any one or more of
Analog 1, Analog 2, Analog 3, Analog 4, Analog 5, Analog 6, Analog
7, Analog 8, Analog 9, Analog 10, Analog 11, or Analog 12, or any
combination thereof. In some embodiments, the compound is Analog 4.
In some embodiments, the compound is Analog 11.
[0180] In some embodiments, the compound(s) of Formula I or a
pharmaceutically acceptable salt thereof are a component of a
pharmaceutical composition further comprising a pharmaceutically
acceptable carrier. In some embodiments, the compound(s) of Formula
Ia or a pharmaceutically acceptable salt thereof are a component of
a pharmaceutical composition further comprising a pharmaceutically
acceptable carrier. In some embodiments, the compound(s) of Formula
II or a pharmaceutically acceptable salt thereof are a component of
a pharmaceutical composition further comprising a pharmaceutically
acceptable carrier. In some embodiments, the compound(s) of Formula
III or a pharmaceutically acceptable salt thereof are a component
of a pharmaceutical composition further comprising a
pharmaceutically acceptable carrier.
[0181] The pharmaceutical composition my optionally comprise a
neoadjuvant therapeutic agent, a chemotherapeutic agent, an
immunotherapeutic agent, a lysosome inhibitor, or a calpain
inhibitor, or any combination thereof.
[0182] Neoadjuvant therapeutic agents include all forms of
treatment of cancer including, but not limited to, traditional
chemotherapy (i.e., anti-cancer agents or chemotherapeutic agents,
whether they are administered parenterally or orally),
immunotherapy, small molecule enzyme or kinase inhibitors,
intravesical therapies, antibody inhibitors of receptors or
kinases, antibody-drug conjugates, and radiation therapy.
[0183] Examples of chemotherapeutic agents include, without
limitation, methotrexate, taxol, mercaptopurine, thioguanine,
hydroxyurea, cytarabine, mitomycin, cyclophosphamide, ifosfamide,
nitrosourea, cisplatin, carboplatin, oxaliplatin, nedaplatin,
triplatin tetranitrate, phenanthriplatin, picoplatin, satraplatin,
dacarbazine, procarbizine, an etoposide, a campathecin, bleomycin,
doxorubicin, idarubicin, daunorubicin, dactinomycin, distamycin A,
etidium, netropsin, auristatin, amsacrine, prodigiosin, bortexomib,
pibenzimol, tomaymycin, duocarmycin SA, plicamycin, mitoxantrone,
asparaginase, vinblastine, vincristine, MG132, tunicamycin,
oligomycin, vinorelbine, paclitaxel, docetaxel, CPT-11, gleevec,
erlotinib, gefitinib, ibrutinib, crizotinib, ceritinib,
flavopiridol, gemcitabine, lapatinib, navitoclax, sorafenib,
regorafenib, ganetespib, irinotecan, or 5-fluorouracil, or any
combination thereof. In some embodiments, the chemotherapeutic
agent is a combination of agents, such as, for example,
methotrexate/vincristine/doxorubicin/cisplatin (MVAC) or
gemcitabine/cisplatin.
[0184] In some embodiments, the neoadjuvant agent is an
immunotherapeutic agent such as, for example, OPDIVO.RTM.
(nivolumab), KEYTRUDA.RTM. (pembrolizumab), TECENTRIQ.RTM.
(atezolizumab), IMFINZI.RTM. (durvalab), YERVOY.RTM. (ipilumumab),
BAVENCIO.RTM. (avelumab), ERBITUX.RTM. (cetuxumab), AVASTIN.RTM.
(bevacizumab), or HERCEPTIN.RTM. (trastuzumab), or any combination
thereof.
[0185] An example of a lysosome inhibitor is chloroquine.
[0186] Examples of calpain inhibitors include, without limitation,
AK275, MDL28170, PD150606, SJA6017, ABT-705253, or SNJ-1945, or any
combination thereof.
[0187] In some embodiments, the ratio of the compound to the
chemotherapeutic agent, the lysosome inhibitor, the immunotherapy
agent, or the calpain inhibitor in the pharmaceutical compositon is
from about 0.01:1 to about 100:1 w/w.
[0188] The compositions may be prepared to provide from about 0.05
mg to about 500 mg of the compound, or pharmaceutically acceptable
salt thereof. The compositions may comprise from about 1 mg to
about 200 mg of the compound, may comprise from about 10 mg to
about 200 mg of the compound, may comprise from about 10 mg to
about 100 mg of the compound, may comprise from about 50 mg to
about 100 mg of the compound, may comprise from about 20 mg to
about 400 mg of the compound, may comprise from about 100 mg to
about 300 mg of the compound, and may comprise from about 50 mg to
about 250 mg of the compound, or pharmaceutically acceptable salt
thereof.
[0189] The pharmaceutical compositons described herein can be
administered to a patient in need thereof in an oral formulation,
an intravenous formulation, a topical formulation, an
intraperitoneal formulation, an intrapleural formulation, an
intravesical formulation, or an intrathecal formulation. The
compositions may be formulated in a suitable controlled-release
vehicle, with an adjuvant, or as a depot formulation.
[0190] Preparations for parenteral administration include sterile
solutions ready for injection, sterile dry soluble products ready
to be combined with a solvent just prior to use, including
hypodermic tablets, sterile suspensions ready for injection,
sterile dry insoluble products ready to be combined with a vehicle
just prior to use and sterile emulsions.
[0191] Solid dosage forms include tablets, pills, powders, bulk
powders, capsules, granules, and combinations thereof. Solid dosage
forms may be prepared as compressed, chewable lozenges and tablets
which may be enteric-coated, sugar coated or film-coated. Solid
dosage forms may be hard or encased in soft gelatin, and granules
and powders may be provided in non-effervescent or effervescent
form. Solid dosage forms may be prepared for dissolution or
suspension in a liquid or semi-liquid vehicle prior to
administration. Solid dosage forms may be prepared for immediate
release, controlled release, or any combination thereof. Controlled
release includes, but is not limited to delayed release, sustained
release, timed pulsatile release, and location-specific pulsatile
release, and combinations thereof.
[0192] Liquid dosage forms include aqueous solutions, emulsions,
suspensions, solutions and/or suspensions reconstituted from
non-effervescent granules and effervescent preparations
reconstituted from effervescent granules. Aqueous solutions
include, for example, elixirs and syrups. Emulsions may be oil-in
water or water-in-oil emulsions.
[0193] In some embodiments, the oral formulation is a pill, tablet,
capsule, cachet, gel-cap, pellet, powder, granule, or liquid.
[0194] Pharmaceutically acceptable excipients utilized in solid
dosage forms include coatings, binders, lubricants, diluents,
disintegrating agents, coloring agents, flavoring agents,
preservatives, sweeteners, and wetting agents. Enteric-coated
tablets, due to their enteric-coating, resist the action of stomach
acid and dissolve or disintegrate in the neutral or alkaline
intestines. Other examples of coatings include sugar coatings and
polymer coatings. Sweetening agents are especially useful in the
formation of chewable tablets and lozenges. Pharmaceutically
acceptable excipients used in liquid dosage forms includes
solvents, suspending agents, dispersing agents, emulsifying agents,
surfactants, emollients, coloring agents, flavoring agents,
preservatives, and sweeteners.
[0195] Non-limiting examples of binders include glucose solution,
acacia mucilage, gelatin solution, sucrose and starch paste.
Non-limiting examples of lubricants include talc, starch, magnesium
or calcium stearate, lycopodium and stearic acid. Non-limiting
examples of diluents include lactose, sucrose, starch, kaolin,
salt, mannitol and dicalcium phosphate. Non-limiting examples of
disintegrating agents include corn starch, potato starch,
bentonite, methylcellulose, agar and carboxymethylcellulose.
Non-limiting examples of emulsifying agents include gelatin,
acacia, tragacanth, bentonite, and surfactants such as
polyoxyethylene sorbitan monooleate. Non-limiting examples of
suspending agents include sodium carboxymethylcellulose, pectin,
tragacanth, veegum and acacia.
[0196] Non-limiting examples of coloring agents include any of the
approved certified water soluble FD and C dyes, mixtures thereof,
and water insoluble FD and D dyes suspended on alumina hydrate.
Non-limiting examples of sweetening agents include dextrose,
sucrose, fructose, lactose, mannitol and artificial sweetening
agents such as saccharin, aspartame, sucralose, acelsulfame
potassium, and other artificial sweeteners. Non-limiting examples
of flavoring agents include synthetic flavors and natural flavors
extracted from plants such as fruits and mints, and synthetic
blends of compounds which produce a pleasant sensation.
Non-limiting examples of wetting agents include propylene glycol
monostearate, sorbitan monooleate, diethylene glycol monolaurate
and polyoxyethylene laural ether. Non-limiting examples of
enteric-coatings include fatty acids, fats, waxes, shellac,
ammoniated shellac and cellulose acetate phthalates. Non-limiting
examples of film coatings include hydroxyethylcellulose, sodium
carboxymethylcellulose, polyethylene glycol 4000 and cellulose
acetate phthalate. Non-limiting examples of preservatives include
glycerin, methyl and propylparaben, ethylparaben, butylparaben,
isobutylparaben, isopropylparaben, benzylparaben, citrate, benzoic
acid, sodium benzoate and alcohol.
[0197] Elixirs include clear, sweetened, hydroalcoholic
preparations. Pharmaceutically acceptable carriers used in elixirs
include solvents. Syrups include concentrated aqueous solutions of
a sugar, for example, sucrose, and may contain a preservative. An
emulsion is a two-phase system in which one liquid is dispersed
throughout another liquid. Pharmaceutically acceptable carriers
used in emulsions may include emulsifying agents and preservatives.
Suspensions may use pharmaceutically acceptable suspending agents
and preservatives. Pharmaceutically acceptable substances used in
non-effervescent granules, to be reconstituted into a liquid oral
dosage form, include diluents, sweeteners and wetting agents.
Pharmaceutically acceptable substance used in effervescent
granules, to be reconstituted into a liquid oral dosage form,
include organic acids and a source of carbon dioxide. Sources of
carbon dioxide include sodium bicarbonate and sodium carbonate.
Coloring and flavoring agents may be used in all such dosage
forms.
[0198] Additional excipients that may be included in any dosage
forms include, but are not limited to antimicrobial agents,
isotonic agents, buffers, antioxidants, local anesthetic agents,
sequestering or chelating agents, analgesic agents, antiemetic
agents, and other agents to enhance selected characteristics of the
formulation.
[0199] Antimicrobial agents may be cidal or static, and may be
antimicrobial, antifungal, antiparasitic, or antiviral.
Non-limiting examples of commonly used antimicrobial agents include
phenols or cresols, mercurials, benzyl alcohol, chlorobutanol,
methyl and propyl p-hydroxybenzoic acid esters, thimerosal,
benzalkonium chloride and benzethonium chloride. Acidic or basic pH
may be used for antimicrobial effects in some aspects. Non-limiting
examples of isotonic agents include sodium chloride and dextrose.
Non-limiting examples of buffers include phosphate and citrate
buffers. A non-limiting example of a chelating agent for metal ions
is EDTA.
[0200] The present disclosure also provides methods for treating a
cancer in a mammal. The methods comprise administering to the
mammal in need thereof a compound of Formula I:
##STR00014##
or a pharmaceutically acceptable salt thereof, wherein:
[0201] R.sub.1 is hydrogen or a substituted or unsubstituted
haloalkyl group; and
[0202] each R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and
R.sub.7 is, independently, hydrogen, halogen, hydroxyl, cyano,
substituted or unsubstituted alkyl, substituted or unsubstituted
alkenyl, substituted or unsubstituted alkoxy, substituted or
unsubstituted aryl, substituted or unsubstituted heteroaryl,
substituted or unsubstituted phosphate, substituted or
unsubstituted phosphoramidate, substituted or unsubstituted amine,
substituted or unsubstituted alkylamino, substituted or
unsubstituted acylamino, substituted or unsubstituted aminoalkoxy,
or substituted or unsubstituted alkylthio.
[0203] In some embodiments, each of R.sub.2, R.sub.3, R.sub.4,
R.sub.5, R.sub.6, and R.sub.7 is, independently, hydrogen, halogen,
hydroxyl, substituted or unsubstituted alkyl, or substituted or
unsubstituted alkoxy. In some embodiments, each of R.sub.2,
R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is, independently,
hydrogen, halogen, or substituted or unsubstituted alkyl. In some
embodiments, each of R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6,
and R.sub.7 is hydrogen. In some embodiments, each of R.sub.2,
R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is not
hydrogen.
[0204] In some embodiments, R.sub.1 is --CF.sub.3,
--C.sub.2F.sub.5, --CHF.sub.2, --CCl.sub.3, --CHCl.sub.2,
--C.sub.2Cl.sub.5, or --CH.sub.2CF.sub.3.
[0205] In some embodiments, R.sub.1 is a substituted or
unsubstituted haloalkyl group; and each of R.sub.2, R.sub.3,
R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is independently, hydrogen,
halogen, hydroxyl, substituted or unsubstituted alkyl, or
substituted or unsubstituted alkoxy.
[0206] In some embodiments, R.sub.1 is --CF.sub.3,
--C.sub.2F.sub.5, --CHF.sub.2, --CCl.sub.3, --CHCl.sub.2,
--C.sub.2Cl.sub.5, or --CH.sub.2CF.sub.3; and each of R.sub.2,
R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is independently,
hydrogen, halogen, or substituted or unsubstituted alkyl.
[0207] In some embodiments, R.sub.1 is --CF.sub.3; and each of
R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is
hydrogen.
[0208] In some embodiments, R.sub.1 is --CF.sub.3; and each of
R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is not
hydrogen.
[0209] The present disclosure also provides methods of treating a
cancer in a mammal comprising administering to the mammal in need
thereof a compound of Formula Ia:
##STR00015##
or a pharmaceutically acceptable salt thereof, wherein:
[0210] R.sub.1 is hydrogen or a substituted or unsubstituted
alkyl;
[0211] each R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and
R.sub.7 is, independently, hydrogen, halogen, hydroxyl, cyano,
substituted or unsubstituted alkyl, substituted or unsubstituted
alkenyl, substituted or unsubstituted haloalkyl, substituted or
unsubstituted alkoxy, substituted or unsubstituted aryl,
substituted or unsubstituted heteroaryl, substituted or
unsubstituted phosphate, substituted or unsubstituted
phosphoramidate, substituted or unsubstituted amine, substituted or
unsubstituted alkylamino, substituted or unsubstituted acylamino,
substituted or unsubstituted aminoalkoxy, or substituted or
unsubstituted alkylthio; and
[0212] n is an integer from 0 to 5.
[0213] In some embodiments, the compound of Formula Ia is not
##STR00016##
[0214] In some embodiments, R.sub.1 is hydrogen, unsubstituted
alkyl, --CF.sub.3, --C.sub.2F.sub.5, --CHF.sub.2, --CCl.sub.3,
--CHCl.sub.2, --C.sub.2Cl.sub.5, or --CH.sub.2CF.sub.3. In some
embodiments, R.sub.1 is hydrogen or unsubstituted alkyl. In some
embodiments, R.sub.1 is hydrogen or methyl.
[0215] In some embodiments, each R.sub.2, R.sub.3, R.sub.4,
R.sub.5, R.sub.6, and R.sub.7 is, independently, hydrogen, halogen,
hydroxyl, cyano, substituted or unsubstituted alkyl, substituted or
unsubstituted haloalkyl, or substituted or unsubstituted alkoxy. In
some embodiments, each R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6,
and R.sub.7 is, independently, hydrogen, halogen, substituted or
unsubstituted alkyl, substituted or unsubstituted haloalkyl, or
substituted or unsubstituted alkoxy. In some embodiments, each
R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is,
independently, hydrogen, halogen, unsubstituted alkyl,
unsubstituted haloalkyl, or unsubstituted alkoxy. In some
embodiments, each R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and
R.sub.7 is, independently, hydrogen, F, Cl, methyl, --CF.sub.3, or
methoxy.
[0216] In some embodiments, n is an integer from 0 to 3. In some
embodiments, n is an integer from 0 to 2. In some embodiments, n is
0 or 1.
[0217] In some embodiments, R.sub.1 is hydrogen, unsubstituted
alkyl, --CF.sub.3, --C.sub.2F.sub.5, --CHF.sub.2, --CCl.sub.3,
--CHCl.sub.2, --C.sub.2Cl.sub.5, or --CH.sub.2CF.sub.3; n is an
integer from 0 to 3; and each R.sub.2, R.sub.3, R.sub.4, R.sub.5,
R.sub.6, and R.sub.7 is, independently, hydrogen, halogen,
hydroxyl, cyano, substituted or unsubstituted alkyl, substituted or
unsubstituted haloalkyl, or substituted or unsubstituted
alkoxy.
[0218] In some embodiments, R.sub.1 is hydrogen or unsubstituted
alkyl; n is an integer from 0 to 2; and each R.sub.2, R.sub.3,
R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is, independently, hydrogen,
halogen, substituted or unsubstituted alkyl, substituted or
unsubstituted haloalkyl, or substituted or unsubstituted
alkoxy.
[0219] In some embodiments, R.sub.1 is hydrogen or methyl; n is 0
or 1; and each R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and
R.sub.7 is, independently, hydrogen, halogen, unsubstituted alkyl,
unsubstituted haloalkyl, or unsubstituted alkoxy.
[0220] In some embodiments, R.sub.1 is hydrogen or methyl; n is 0
or 1; and each R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and
R.sub.7 is, independently, hydrogen, F, Cl, methyl, --CF.sub.3, or
methoxy.
[0221] In some embodiments, each R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is hydrogen; and n is 0.
[0222] In some embodiments, each R.sub.1, R.sub.2, R.sub.3,
R.sub.5, and R.sub.7 is hydrogen; each R.sub.4 and R.sub.6 is
methyl; and n is 0.
[0223] In some embodiments, each R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.6, and R.sub.7 is hydrogen; R.sub.5 is methoxy; and
n is 0.
[0224] In some embodiments, each R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.5, and R.sub.7 is hydrogen; R.sub.6 is --CF.sub.3;
and n is 0.
[0225] In some embodiments, each R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.6, and R.sub.7 is hydrogen; R.sub.5 is fluorine; and
n is 1.
[0226] In some embodiments, each R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.6, and R.sub.7 is hydrogen; R.sub.5 is methoxy; and
n is 1.
[0227] In some embodiments, each R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.6, and R.sub.7 is hydrogen; R.sub.5 is chlorine; and
n is 1.
[0228] In some embodiments, R.sub.1 is methyl; each R.sub.2,
R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is hydrogen; and n
is 0.
[0229] In some embodiments, R.sub.1 is methyl; each R.sub.2,
R.sub.3, R.sub.4, and R.sub.7 is hydrogen; R.sub.5 and R.sub.6 are
methyl; and n is 0.
[0230] The present disclosure also provides methods of treating a
cancer in a mammal comprising administering to the mammal in need
thereof a compound of Formula II:
##STR00017##
or a pharmaceutically acceptable salt thereof, wherein:
[0231] R.sub.1 is hydrogen or a substituted or unsubstituted alkyl
group;
[0232] each R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and
R.sub.7 is, independently, hydrogen, halogen, hydroxyl, substituted
or unsubstituted alkyl, substituted or unsubstituted alkoxy,
substituted or unsubstituted aryl, or substituted or unsubstituted
amine;
[0233] X is sulfur, oxygen, or --NH; and
[0234] n is an integer from 0 to 5.
[0235] In some embodiments, R.sub.1 is hydrogen or an unsubstituted
alkyl. In some embodiments, R.sub.1 is hydrogen or methyl. In some
embodiments, R.sub.1 is hydrogen.
[0236] In some embodiments, each R.sub.2, R.sub.3, R.sub.4,
R.sub.5, R.sub.6, and R.sub.7 is, independently, hydrogen, halogen,
hydroxyl, substituted or unsubstituted alkyl, substituted or
unsubstituted alkoxy, substituted or unsubstituted aryl, or
substituted or unsubstituted amine. In some embodiments, each
R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is,
independently, hydrogen, halogen, or substituted or unsubstituted
alkyl. In some embodiments, each R.sub.2, R.sub.3, R.sub.4,
R.sub.5, R.sub.6, and R.sub.7 is, independently, hydrogen, halogen,
or unsubstituted alkyl. In some embodiments, each R.sub.2, R.sub.3,
R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is, independently, hydrogen,
halogen, or methyl.
[0237] In some embodiments, X is sulfur or oxygen. In some
embodiments, X is sulfur.
[0238] In some embodiments, n is an integer from 0 to 3. In some
embodiments, n is an integer from 0 to 2. In some embodiments, n is
1 or 2.
[0239] In some embodiments, R.sub.1 is hydrogen or an unsubstituted
alkyl; each R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and
R.sub.7 is, independently, hydrogen, halogen, hydroxyl, substituted
or unsubstituted alkyl, substituted or unsubstituted alkoxy,
substituted or unsubstituted aryl, or substituted or unsubstituted
amine; X is sulfur or oxygen; and n is an integer from 0 to 3.
[0240] In some embodiments, R.sub.1 is hydrogen or methyl; each
R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is,
independently, hydrogen, halogen, or substituted or unsubstituted
alkyl; X is sulfur or oxygen; and n is an integer from 0 to 2.
[0241] In some embodiments, R.sub.1 is hydrogen; each R.sub.2,
R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is, independently,
hydrogen, halogen, or unsubstituted alkyl; X is sulfur; and n is 1
or 2.
[0242] In some embodiments, R.sub.1 is hydrogen; each R.sub.2,
R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is, independently,
hydrogen, halogen, or methyl; X is sulfur; and n is 1 or 2.
[0243] In some embodiments, each R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.6, and R.sub.7 is hydrogen; R.sub.5 is methyl; X is
sulfur; and n is 2.
[0244] In some embodiments, each R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.6, and R.sub.7 is hydrogen; R.sub.5 is chlorine; X
is sulfur; and n is 2.
[0245] The present disclosure also provides methods of treating a
cancer in a mammal comprising administering to the mammal in need
thereof a compound of Formula III:
##STR00018##
or a pharmaceutically acceptable salt thereof, wherein:
[0246] R.sub.1 and R.sub.2 are, independently, hydrogen or a
substituted or unsubstituted alkyl;
[0247] R.sub.3 is a substituted or unsubstituted heteroaryl;
and
[0248] n is an integer from 0 to 5.
[0249] In some embodiments, R.sub.1 and R.sub.2 are, independently,
hydrogen or an unsubstituted alkyl. In some embodiments, R.sub.1
and R.sub.2 are, independently, hydrogen or methyl. In some
embodiments, R.sub.1 and R.sub.2 are hydrogen.
[0250] In some embodiments, n is an integer from 0 to 3. In some
embodiments, n is an integer from 0 to 2. In some embodiments, n is
1.
[0251] In some embodiments, R.sub.3 is an unsubstituted heteroaryl.
In some embodiments, R.sub.3 is unsubstituted heteroaryl having
from 3 to 10 ring-forming atoms. In some embodiments, R.sub.3 is
unsubstituted heteroaryl having from 3 to 6 ring-forming atoms. In
some embodiments, R.sub.3 is unsubstituted heteroaryl having from 3
to 5 ring-forming atoms. In some embodiments, R.sub.3 is pyridyl,
pyrimidinyl, pyrazinyl, pyridazinyl, pyridinyl, triazinyl, furyl,
quinolyl, isoquinolyl, thienyl, imidazolyl, thiazolyl, indolyl,
pyrryl, oxazolyl, benzofuryl, benzothienyl, pyrazolyl,
benzthiazolyl, isoxazolyl, triazolyl, tetrazolyl, indazolyl,
isothiazolyl, 1,2,4-thiadiazolyl, benzothienyl, purinyl,
carbazolyl, isoxazolyl, benzimidazolyl, indolinyl, pyranyl,
pyrazolyl, triazolyl, oxadiazolyl, thianthrenyl, indolizinyl,
isoindolyl, isobenzofuranyl, pyrrolyl, benzoxazolyl, xanthenyl,
2H-pyrrolyl, 3H-indolyl, 4H-quinolizinyl, phthalazinyl, acridinyl,
naphthyridinyl, quinazolinyl, phenanthridinyl, perimidinyl,
phenanthrolinyl, phenazinyl, isothiazolyl, phenothiazinyl,
isoxazolyl, or furazanyl. In some embodiments, R.sub.3 is pyridyl,
pyrimidinyl, pyrazinyl, pyridazinyl, pyridinyl, triazinyl, furyl,
quinolyl, isoquinolyl, thienyl, imidazolyl, thiazolyl, indolyl,
pyrryl, oxazolyl, purinyl, benzimidazolyl, indolinyl, pyranyl,
pyrazolyl, triazolyl, oxadiazolyl, thianthrenyl, or pyrrolyl. In
some embodiments, R.sub.3 is 2-thienyl or 3-thienyl.
[0252] In some embodiments, R.sub.1 and R.sub.2 are, independently,
hydrogen or an unsubstituted alkyl; n is an integer from 0 to 3;
and R.sub.3 is an unsubstituted heteroaryl.
[0253] In some embodiments, R.sub.1 and R.sub.2 are, independently,
hydrogen or an unsubstituted alkyl; n is an integer from 0 to 3;
and R.sub.3 is unsubstituted heteroaryl having from 3 to 10
ring-forming atoms.
[0254] In some embodiments, R.sub.1 and R.sub.2 are, independently,
hydrogen or methyl; n is an integer from 0 to 2; and R.sub.3 is
unsubstituted heteroaryl having from 3 to 6 ring-forming atoms.
[0255] In some embodiments, R.sub.1 and R.sub.2 are, independently,
hydrogen or methyl; n is an integer from 0 to 2; and R.sub.3 is
unsubstituted heteroaryl having from 3 to 5 ring-forming atoms.
[0256] In some embodiments, R.sub.1 and R.sub.2 are hydrogen; n is
an integer from 0 to 2; and R.sub.3 is pyridyl, pyrimidinyl,
pyrazinyl, pyridazinyl, pyridinyl, triazinyl, furyl, quinolyl,
isoquinolyl, thienyl, imidazolyl, thiazolyl, indolyl, pyrryl,
oxazolyl, benzofuryl, benzothienyl, pyrazolyl, benzthiazolyl,
isoxazolyl, triazolyl, tetrazolyl, indazolyl, isothiazolyl,
1,2,4-thiadiazolyl, benzothienyl, purinyl, carbazolyl, isoxazolyl,
benzimidazolyl, indolinyl, pyranyl, pyrazolyl, triazolyl,
oxadiazolyl, thianthrenyl, indolizinyl, isoindolyl,
isobenzofuranyl, pyrrolyl, benzoxazolyl, xanthenyl, 2H-pyrrolyl,
3H-indolyl, 4H-quinolizinyl, phthalazinyl, acridinyl,
naphthyridinyl, quinazolinyl, phenanthridinyl, perimidinyl,
phenanthrolinyl, phenazinyl, isothiazolyl, phenothiazinyl,
isoxazolyl, or furazanyl.
[0257] In some embodiments, R.sub.1 and R.sub.2 are hydrogen; n is
1; and R.sub.3 is pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl,
pyridinyl, triazinyl, furyl, quinolyl, isoquinolyl, thienyl,
imidazolyl, thiazolyl, indolyl, pyrryl, oxazolyl, purinyl,
benzimidazolyl, indolinyl, pyranyl, pyrazolyl, triazolyl,
oxadiazolyl, thianthrenyl, or pyrrolyl.
[0258] In some embodiments, R.sub.1 and R.sub.2 are hydrogen; n is
1; and R.sub.3 is 2-thienyl or 3-thienyl.
[0259] In some embodiments, R.sub.1 and R.sub.2 are hydrogen;
R.sub.3 is -2-thienyl; and n is 1.
[0260] The present disclosure also provides methods for restoring
the tumor suppressor protein p53 signaling pathway within a tumor
cell of a mammal comprising administering to the mammal in need
thereof a compound of Formula I:
##STR00019##
or a pharmaceutically acceptable salt thereof, wherein:
[0261] R.sub.1 is hydrogen or a substituted or unsubstituted
haloalkyl group; and
[0262] each R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and
R.sub.7 is, independently, hydrogen, halogen, hydroxyl, cyano,
substituted or unsubstituted alkyl, substituted or unsubstituted
alkenyl, substituted or unsubstituted alkoxy, substituted or
unsubstituted aryl, substituted or unsubstituted heteroaryl,
substituted or unsubstituted phosphate, substituted or
unsubstituted phosphoramidate, substituted or unsubstituted amine,
substituted or unsubstituted alkylamino, substituted or
unsubstituted acylamino, substituted or unsubstituted aminoalkoxy,
or substituted or unsubstituted alkylthio.
[0263] In some embodiments, each of R.sub.2, R.sub.3, R.sub.4,
R.sub.5, R.sub.6, and R.sub.7 is, independently, hydrogen, halogen,
hydroxyl, substituted or unsubstituted alkyl, or substituted or
unsubstituted alkoxy. In some embodiments, each of R.sub.2,
R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is, independently,
hydrogen, halogen, or substituted or unsubstituted alkyl. In some
embodiments, each of R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6,
and R.sub.7 is hydrogen. In some embodiments, each of R.sub.2,
R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is not
hydrogen.
[0264] In some embodiments, R.sub.1 is --CF.sub.3,
--C.sub.2F.sub.5, --CHF.sub.2, --CCl.sub.3, --CHCl.sub.2,
--C.sub.2Cl.sub.5, or --CH.sub.2CF.sub.3.
[0265] In some embodiments, R.sub.1 is a substituted or
unsubstituted haloalkyl group; and each of R.sub.2, R.sub.3,
R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is independently, hydrogen,
halogen, hydroxyl, substituted or unsubstituted alkyl, or
substituted or unsubstituted alkoxy.
[0266] In some embodiments, R.sub.1 is --CF.sub.3,
--C.sub.2F.sub.5, --CHF.sub.2, --CCl.sub.3, --CHCl.sub.2,
--C.sub.2Cl.sub.5, or --CH.sub.2CF.sub.3; and each of R.sub.2,
R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is independently,
hydrogen, halogen, or substituted or unsubstituted alkyl.
[0267] In some embodiments, R.sub.1 is --CF.sub.3; and each of
R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is
hydrogen.
[0268] In some embodiments, R.sub.1 is --CF.sub.3; and each of
R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is not
hydrogen.
[0269] The present disclosure also provides methods of restoring
the tumor suppressor protein p53 signaling pathway within a tumor
cell of a mammal comprising administering to the mammal in need
thereof a compound of Formula Ia:
##STR00020##
or a pharmaceutically acceptable salt thereof, wherein:
[0270] R.sub.1 is hydrogen or a substituted or unsubstituted
alkyl;
[0271] each R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and
R.sub.7 is, independently, hydrogen, halogen, hydroxyl, cyano,
substituted or unsubstituted alkyl, substituted or unsubstituted
alkenyl, substituted or unsubstituted haloalkyl, substituted or
unsubstituted alkoxy, substituted or unsubstituted aryl,
substituted or unsubstituted heteroaryl, substituted or
unsubstituted phosphate, substituted or unsubstituted
phosphoramidate, substituted or unsubstituted amine, substituted or
unsubstituted alkylamino, substituted or unsubstituted acylamino,
substituted or unsubstituted aminoalkoxy, or substituted or
unsubstituted alkylthio; and
[0272] n is an integer from 0 to 5.
[0273] In some embodiments, the compound of Formula Ia is not
##STR00021##
[0274] In some embodiments, R.sub.1 is hydrogen, unsubstituted
alkyl, --CF.sub.3, --C.sub.2F.sub.5, --CHF.sub.2, --CCl.sub.3,
--CHCl.sub.2, --C.sub.2Cl.sub.5, or --CH.sub.2CF.sub.3. In some
embodiments, R.sub.1 is hydrogen or unsubstituted alkyl. In some
embodiments, R.sub.1 is hydrogen or methyl.
[0275] In some embodiments, each R.sub.2, R.sub.3, R.sub.4,
R.sub.5, R.sub.6, and R.sub.7 is, independently, hydrogen, halogen,
hydroxyl, cyano, substituted or unsubstituted alkyl, substituted or
unsubstituted haloalkyl, or substituted or unsubstituted alkoxy. In
some embodiments, each R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6,
and R.sub.7 is, independently, hydrogen, halogen, substituted or
unsubstituted alkyl, substituted or unsubstituted haloalkyl, or
substituted or unsubstituted alkoxy. In some embodiments, each
R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is,
independently, hydrogen, halogen, unsubstituted alkyl,
unsubstituted haloalkyl, or unsubstituted alkoxy. In some
embodiments, each R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and
R.sub.7 is, independently, hydrogen, F, Cl, methyl, --CF.sub.3, or
methoxy.
[0276] In some embodiments, n is an integer from 0 to 3. In some
embodiments, n is an integer from 0 to 2. In some embodiments, n is
0 or 1.
[0277] In some embodiments, R.sub.1 is hydrogen, unsubstituted
alkyl, --CF.sub.3, --C.sub.2F.sub.5, --CHF.sub.2, --CCl.sub.3,
--CHCl.sub.2, --C.sub.2Cl.sub.5, or --CH.sub.2CF.sub.3; n is an
integer from 0 to 3; and each R.sub.2, R.sub.3, R.sub.4, R.sub.5,
R.sub.6, and R.sub.7 is, independently, hydrogen, halogen,
hydroxyl, cyano, substituted or unsubstituted alkyl, substituted or
unsubstituted haloalkyl, or substituted or unsubstituted
alkoxy.
[0278] In some embodiments, R.sub.1 is hydrogen or unsubstituted
alkyl; n is an integer from 0 to 2; and each R.sub.2, R.sub.3,
R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is, independently, hydrogen,
halogen, substituted or unsubstituted alkyl, substituted or
unsubstituted haloalkyl, or substituted or unsubstituted
alkoxy.
[0279] In some embodiments, R.sub.1 is hydrogen or methyl; n is 0
or 1; and each R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and
R.sub.7 is, independently, hydrogen, halogen, unsubstituted alkyl,
unsubstituted haloalkyl, or unsubstituted alkoxy.
[0280] In some embodiments, R.sub.1 is hydrogen or methyl; n is 0
or 1; and each R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and
R.sub.7 is, independently, hydrogen, F, Cl, methyl, --CF.sub.3, or
methoxy.
[0281] In some embodiments, each R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is hydrogen; and n is 0.
[0282] In some embodiments, each R.sub.1, R.sub.2, R.sub.3,
R.sub.5, and R.sub.7 is hydrogen; each R.sub.4 and R.sub.6 is
methyl; and n is 0.
[0283] In some embodiments, each R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.6, and R.sub.7 is hydrogen; R.sub.5 is methoxy; and
n is 0.
[0284] In some embodiments, each R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.5, and R.sub.7 is hydrogen; R.sub.6 is --CF.sub.3;
and n is 0.
[0285] In some embodiments, each R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.6, and R.sub.7 is hydrogen; R.sub.5 is fluorine; and
n is 1.
[0286] In some embodiments, each R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.6, and R.sub.7 is hydrogen; R.sub.5 is methoxy; and
n is 1.
[0287] In some embodiments, each R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.6, and R.sub.7 is hydrogen; R.sub.5 is chlorine; and
n is 1.
[0288] In some embodiments, R.sub.1 is methyl; each R.sub.2,
R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is hydrogen; and n
is 0.
[0289] In some embodiments, R.sub.1 is methyl; each R.sub.2,
R.sub.3, R.sub.4, and R.sub.7 is hydrogen; R.sub.5 and R.sub.6 are
methyl; and n is 0.
[0290] The present disclosure also provides methods of restoring
the tumor suppressor protein p53 signaling pathway within a tumor
cell of a mammal comprising administering to the mammal in need
thereof a compound of Formula II:
##STR00022##
or a pharmaceutically acceptable salt thereof, wherein:
[0291] R.sub.1 is hydrogen or a substituted or unsubstituted alkyl
group;
[0292] each R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and
R.sub.7 is, independently, hydrogen, halogen, hydroxyl, substituted
or unsubstituted alkyl, substituted or unsubstituted alkoxy,
substituted or unsubstituted aryl, or substituted or unsubstituted
amine;
[0293] X is sulfur, oxygen, or --NH; and
[0294] n is an integer from 0 to 5.
[0295] In some embodiments, R.sub.1 is hydrogen or an unsubstituted
alkyl. In some embodiments, R.sub.1 is hydrogen or methyl. In some
embodiments, R.sub.1 is hydrogen.
[0296] In some embodiments, each R.sub.2, R.sub.3, R.sub.4,
R.sub.5, R.sub.6, and R.sub.7 is, independently, hydrogen, halogen,
hydroxyl, substituted or unsubstituted alkyl, substituted or
unsubstituted alkoxy, substituted or unsubstituted aryl, or
substituted or unsubstituted amine. In some embodiments, each
R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is,
independently, hydrogen, halogen, or substituted or unsubstituted
alkyl. In some embodiments, each R.sub.2, R.sub.3, R.sub.4,
R.sub.5, R.sub.6, and R.sub.7 is, independently, hydrogen, halogen,
or unsubstituted alkyl. In some embodiments, each R.sub.2, R.sub.3,
R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is, independently, hydrogen,
halogen, or methyl.
[0297] In some embodiments, X is sulfur or oxygen. In some
embodiments, X is sulfur.
[0298] In some embodiments, n is an integer from 0 to 3. In some
embodiments, n is an integer from 0 to 2. In some embodiments, n is
1 or 2.
[0299] In some embodiments, R.sub.1 is hydrogen or an unsubstituted
alkyl; each R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and
R.sub.7 is, independently, hydrogen, halogen, hydroxyl, substituted
or unsubstituted alkyl, substituted or unsubstituted alkoxy,
substituted or unsubstituted aryl, or substituted or unsubstituted
amine; X is sulfur or oxygen; and n is an integer from 0 to 3.
[0300] In some embodiments, R.sub.1 is hydrogen or methyl; each
R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is,
independently, hydrogen, halogen, or substituted or unsubstituted
alkyl; X is sulfur or oxygen; and n is an integer from 0 to 2.
[0301] In some embodiments, R.sub.1 is hydrogen; each R.sub.2,
R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is, independently,
hydrogen, halogen, or unsubstituted alkyl; X is sulfur; and n is 1
or 2.
[0302] In some embodiments, R.sub.1 is hydrogen; each R.sub.2,
R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is, independently,
hydrogen, halogen, or methyl; X is sulfur; and n is 1 or 2.
[0303] In some embodiments, each R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.6, and R.sub.7 is hydrogen; R.sub.5 is methyl; X is
sulfur; and n is 2.
[0304] In some embodiments, each R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.6, and R.sub.7 is hydrogen; R.sub.5 is chlorine; X
is sulfur; and n is 2.
[0305] The present disclosure also provides methods of restoring
the tumor suppressor protein p53 signaling pathway within a tumor
cell of a mammal comprising administering to the mammal in need
thereof a compound of Formula III:
##STR00023##
or a pharmaceutically acceptable salt thereof, wherein:
[0306] R.sub.1 and R.sub.2 are, independently, hydrogen or a
substituted or unsubstituted alkyl;
[0307] R.sub.3 is a substituted or unsubstituted heteroaryl;
and
[0308] n is an integer from 0 to 5.
[0309] In some embodiments, R.sub.1 and R.sub.2 are, independently,
hydrogen or an unsubstituted alkyl. In some embodiments, R.sub.1
and R.sub.2 are, independently, hydrogen or methyl. In some
embodiments, R.sub.1 and R.sub.2 are hydrogen.
[0310] In some embodiments, n is an integer from 0 to 3. In some
embodiments, n is an integer from 0 to 2. In some embodiments, n is
1.
[0311] In some embodiments, R.sub.3 is an unsubstituted heteroaryl.
In some embodiments, R.sub.3 is unsubstituted heteroaryl having
from 3 to 10 ring-forming atoms. In some embodiments, R.sub.3 is
unsubstituted heteroaryl having from 3 to 6 ring-forming atoms. In
some embodiments, R.sub.3 is unsubstituted heteroaryl having from 3
to 5 ring-forming atoms. In some embodiments, R.sub.3 is pyridyl,
pyrimidinyl, pyrazinyl, pyridazinyl, pyridinyl, triazinyl, furyl,
quinolyl, isoquinolyl, thienyl, imidazolyl, thiazolyl, indolyl,
pyrryl, oxazolyl, benzofuryl, benzothienyl, pyrazolyl,
benzthiazolyl, isoxazolyl, triazolyl, tetrazolyl, indazolyl,
isothiazolyl, 1,2,4-thiadiazolyl, benzothienyl, purinyl,
carbazolyl, isoxazolyl, benzimidazolyl, indolinyl, pyranyl,
pyrazolyl, triazolyl, oxadiazolyl, thianthrenyl, indolizinyl,
isoindolyl, isobenzofuranyl, pyrrolyl, benzoxazolyl, xanthenyl,
2H-pyrrolyl, 3H-indolyl, 4H-quinolizinyl, phthalazinyl, acridinyl,
naphthyridinyl, quinazolinyl, phenanthridinyl, perimidinyl,
phenanthrolinyl, phenazinyl, isothiazolyl, phenothiazinyl,
isoxazolyl, or furazanyl. In some embodiments, R.sub.3 is pyridyl,
pyrimidinyl, pyrazinyl, pyridazinyl, pyridinyl, triazinyl, furyl,
quinolyl, isoquinolyl, thienyl, imidazolyl, thiazolyl, indolyl,
pyrryl, oxazolyl, purinyl, benzimidazolyl, indolinyl, pyranyl,
pyrazolyl, triazolyl, oxadiazolyl, thianthrenyl, or pyrrolyl. In
some embodiments, R.sub.3 is 2-thienyl or 3-thienyl.
[0312] In some embodiments, R.sub.1 and R.sub.2 are, independently,
hydrogen or an unsubstituted alkyl; n is an integer from 0 to 3;
and R.sub.3 is an unsubstituted heteroaryl.
[0313] In some embodiments, R.sub.1 and R.sub.2 are, independently,
hydrogen or an unsubstituted alkyl; n is an integer from 0 to 3;
and R.sub.3 is unsubstituted heteroaryl having from 3 to 10
ring-forming atoms.
[0314] In some embodiments, R.sub.1 and R.sub.2 are, independently,
hydrogen or methyl; n is an integer from 0 to 2; and R.sub.3 is
unsubstituted heteroaryl having from 3 to 6 ring-forming atoms.
[0315] In some embodiments, R.sub.1 and R.sub.2 are, independently,
hydrogen or methyl; n is an integer from 0 to 2; and R.sub.3 is
unsubstituted heteroaryl having from 3 to 5 ring-forming atoms.
[0316] In some embodiments, R.sub.1 and R.sub.2 are hydrogen; n is
an integer from 0 to 2; and R.sub.3 is pyridyl, pyrimidinyl,
pyrazinyl, pyridazinyl, pyridinyl, triazinyl, furyl, quinolyl,
isoquinolyl, thienyl, imidazolyl, thiazolyl, indolyl, pyrryl,
oxazolyl, benzofuryl, benzothienyl, pyrazolyl, benzthiazolyl,
isoxazolyl, triazolyl, tetrazolyl, indazolyl, isothiazolyl,
1,2,4-thiadiazolyl, benzothienyl, purinyl, carbazolyl, isoxazolyl,
benzimidazolyl, indolinyl, pyranyl, pyrazolyl, triazolyl,
oxadiazolyl, thianthrenyl, indolizinyl, isoindolyl,
isobenzofuranyl, pyrrolyl, benzoxazolyl, xanthenyl, 2H-pyrrolyl,
3H-indolyl, 4H-quinolizinyl, phthalazinyl, acridinyl,
naphthyridinyl, quinazolinyl, phenanthridinyl, perimidinyl,
phenanthrolinyl, phenazinyl, isothiazolyl, phenothiazinyl,
isoxazolyl, or furazanyl.
[0317] In some embodiments, R.sub.1 and R.sub.2 are hydrogen; n is
1; and R.sub.3 is pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl,
pyridinyl, triazinyl, furyl, quinolyl, isoquinolyl, thienyl,
imidazolyl, thiazolyl, indolyl, pyrryl, oxazolyl, purinyl,
benzimidazolyl, indolinyl, pyranyl, pyrazolyl, triazolyl,
oxadiazolyl, thianthrenyl, or pyrrolyl.
[0318] In some embodiments, R.sub.1 and R.sub.2 are hydrogen; n is
1; and R.sub.3 is 2-thienyl or 3-thienyl.
[0319] In some embodiments, R.sub.1 and R.sub.2 are hydrogen;
R.sub.3 is -2-thienyl; and n is 1.
[0320] In some embodiments, the cancer is a tumor suppressor
protein p53 mutated associated cancer, a tumor suppressor protein
Rb mutated associated cancer, a tumor suppressor protein NF1
mutated associated cancer, a tumor suppressor protein p16 mutated
associated cancer, a tumor suppressor protein p27 mutated
associated cancer, or a tumor suppressor protein VHL mutated
associated cancer.
[0321] In some embodiments, the tumor suppressor protein p53
mutated associated cancer is breast cancer, pancreatic cancer,
prostate cancer, lung cancer, liver cancer, oesophagus cancer,
stomach cancer, billary tract cancer, head and neck cancer, bladder
cancer, kidney cancer, mesothelioma, thyroid cancer, uterine
cancer, ovarian cancer, brain cancer, lymphoma, myeloma, leukemia,
or colon cancer.
[0322] In embodiments where the cancer is colon cancer, the colon
cancer is colon adenocarcinoma, a mutated KRAS tumor, a mutated
NRAS tumor, a mutated BRAF tumor, or a tumor with or without
microsatellite instability.
[0323] The amount of compound to be administered may be that amount
which is therapeutically effective. The dosage to be administered
may depend on the characteristics of the subject being treated,
e.g., the particular animal treated, age, weight, health, types of
concurrent treatment, if any, and frequency of treatments, and on
the nature and extent of the disease, condition, or disorder, and
can be easily determined by one skilled in the art (e.g., by the
clinician). The selection of the specific dose regimen can be
selected or adjusted or titrated by the clinician according to
methods known to the clinician to obtain the desired clinical
response. In addition, in vitro or in vivo assays may optionally be
employed to help identify optimal dosage ranges. The precise dose
to be employed in the compositions may also depend on the route of
administration, and should be decided according to the judgment of
the practitioner and each patient's circumstances.
[0324] Suitable dosage ranges for oral administration include, but
are not limited to, from about 0.001 mg/kg body weight to about 200
mg/kg body weight, from about 0.01 mg/kg body weight to about 100
mg/kg body weight, from about 0.01 mg/kg body weight to about 70
mg/kg body weight, from about 0.1 mg/kg body weight to about 50
mg/kg body weight, from 0.5 mg/kg body weight to about 20 mg/kg
body weight, or from about 1 mg/kg body weight to about 10 mg/kg
body weight. In some embodiments, the oral dose is about 5 mg/kg
body weight.
[0325] Suitable dosage ranges for intravenous administration
include, but are not limited to, from about 0.01 mg/kg body weight
to about 500 mg/kg body weight, from about 0.1 mg/kg body weight to
about 100 mg/kg body weight, from about 1 mg/kg body weight to
about 50 mg/kg body weight, or from about 10 mg/kg body weight to
about 35 mg/kg body weight.
[0326] Suitable dosage ranges for other routes of administration
can be calculated based on the forgoing dosages as known by one
skilled in the art. For example, recommended dosages for
intradermal, intramuscular, intraperitoneal, subcutaneous,
epidural, sublingual, intracerebral, transdermal, or inhalation are
in the range from about 0.001 mg/kg body weight to about 200 mg/kg
body weight, from about 0.01 mg/kg body weight to about 100 mg/kg
body weight, from about 0.1 mg/kg body weight to about 50 mg/kg
body weight, or from about 1 mg/kg body weight to about 20 mg/kg
body weight. Effective doses may be extrapolated from dose-response
curves derived from in vitro or animal model test systems. Such
animal models and systems are well known in the art.
[0327] In some embodiments, the amount of the compound of Formula I
administered to the mammal is from about 0.1 mg to about 500 mg. In
some embodiments, the amount of the compound of Formula Ia
administered to the mammal is from about 0.1 mg to about 500 mg. In
some embodiments, the amount of the compound of Formula II
administered to the mammal is from about 0.1 mg to about 500 mg. In
some embodiments, the amount of the compound of Formula III
administered to the mammal is from about 0.1 mg to about 500
mg.
[0328] Co-administration of the compositions described herein with
other means of treatment is also contemplated. For example, a human
in need of cancer treatment may also be administered radiation
therapy, a chemotherapeutic agent, an immunotherapeutic agent, a
lysosome inhibitor, or a calpain inhibitor, or any combination
thereof.
[0329] In some embodiments, the chemotherapeutic agent is
methotrexate, taxol, mercaptopurine, thioguanine, hydroxyurea,
cytarabine, mitomycin, cyclophosphamide, ifosfamide, nitrosourea,
cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin
tetranitrate, phenanthriplatin, picoplatin, satraplatin,
dacarbazine, procarbizine, an etoposide, a campathecin, bleomycin,
doxorubicin, idarubicin, daunorubicin, dactinomycin, distamycin A,
etidium, netropsin, auristatin, amsacrine, prodigiosin, bortexomib,
pibenzimol, tomaymycin, duocarmycin SA, plicamycin, mitoxantrone,
asparaginase, vinblastine, vincristine, MG132, tunicamycin,
oligomycin, vinorelbine, paclitaxel, docetaxel, CPT-11, gleevec,
erlotinib, gefitinib, ibrutinib, crizotinib, ceritinib,
flavopiridol, gemcitabine, lapatinib, navitoclax, sorafenib,
regorafenib, ganetespib, irinotecan, or 5-fluorouracil, or any
combination thereof.
[0330] In some embodiments, a chemotherapeutic agent is also
administered and the chemotherapeutic agent is CPT-11 or
5-fluorouracil.
[0331] In some embodiments, a lysosome inhibitor is also
administered and the lysosome inhibitor is chloroquine.
[0332] In some embodiments, an immunotherapeutic agent is also
administered and the immunotherapeutic agent is OPDIVO.RTM.
(nivolumab), KEYTRUDA.RTM. (pembrolizumab), TECENTRIQ.RTM.
(atezolizumab), IMFINZI.RTM. (durvalab), YERVOY.RTM. (ipilumumab),
BAVENCIO.RTM. (avelumab), ERBITUX.RTM. (cetuxumab), AVASTIN.RTM.
(bevacizumab), or HERCEPTIN.RTM. (trastuzumab), or any combination
thereof.
[0333] In some embodiments, a calpain inhibitor is also
administered and the calpain inhibitor is AK275, MDL28170,
PD150606, SJA6017, ABT-705253, or SNJ-1945, or any combination
thereof.
[0334] In some embodiments, the compound of Formula I, or its
pharmaceutically acceptable salt, is administered prior to the
radiation therapy or prior to administration of the
chemotherapeutic agent, lysosome inhibitor, immunotherapeutic
agent, or calpain inhibitor, or the compound of Formula I is
administered after the radiation therapy or after administration of
the chemotherapeutic agent, lysosome inhibitor, immunotherapeutic
agent, or calpain inhibitor.
[0335] In some embodiments, the compound of Formula I, or its
pharmaceutically acceptable salt, is administered concurrently with
radiation therapy or concurrently with administration of the
chemotherapeutic agent, lysosome inhibitor, immunotherapeutic
agent, or calpain inhibitor.
[0336] In some embodiments, the compound of Formula Ia, or its
pharmaceutically acceptable salt, is administered prior to the
radiation therapy or prior to administration of the
chemotherapeutic agent, lysosome inhibitor, immunotherapeutic
agent, or calpain inhibitor, or the compound of Formula I is
administered after the radiation therapy or after administration of
the chemotherapeutic agent, lysosome inhibitor, immunotherapeutic
agent, or calpain inhibitor.
[0337] In some embodiments, the compound of Formula Ia, or its
pharmaceutically acceptable salt, is administered concurrently with
radiation therapy or concurrently with administration of the
chemotherapeutic agent, lysosome inhibitor, immunotherapeutic
agent, or calpain inhibitor.
[0338] In some embodiments, the compound of Formula II, or its
pharmaceutically acceptable salt, is administered prior to the
radiation therapy or prior to administration of the
chemotherapeutic agent, lysosome inhibitor, immunotherapeutic
agent, or calpain inhibitor, or the compound of Formula I is
administered after the radiation therapy or after administration of
the chemotherapeutic agent, lysosome inhibitor, immunotherapeutic
agent, or calpain inhibitor.
[0339] In some embodiments, the compound of Formula II, or its
pharmaceutically acceptable salt, is administered concurrently with
radiation therapy or concurrently with administration of the
chemotherapeutic agent, lysosome inhibitor, immunotherapeutic
agent, or calpain inhibitor.
[0340] In some embodiments, the compound of Formula III, or its
pharmaceutically acceptable salt, is administered prior to the
radiation therapy or prior to administration of the
chemotherapeutic agent, lysosome inhibitor, immunotherapeutic
agent, or calpain inhibitor, or the compound of Formula I is
administered after the radiation therapy or after administration of
the chemotherapeutic agent, lysosome inhibitor, immunotherapeutic
agent, or calpain inhibitor.
[0341] In some embodiments, the compound of Formula III, or its
pharmaceutically acceptable salt, is administered concurrently with
radiation therapy or concurrently with administration of the
chemotherapeutic agent, lysosome inhibitor, immunotherapeutic
agent, or calpain inhibitor.
[0342] In some embodiments, the compound of Formula I, or its
pharmaceutically acceptable salt, and the chemotherapeutic agent,
lysosome inhibitor, immunotherapeutic agent, or calpain inhibit are
present in the same pharmaceutical composition.
[0343] In some embodiments, the compound of Formula Ia, or its
pharmaceutically acceptable salt, and the chemotherapeutic agent,
lysosome inhibitor, immunotherapeutic agent, or calpain inhibit are
present in the same pharmaceutical composition.
[0344] In some embodiments, the compound of Formula II, or its
pharmaceutically acceptable salt, and the chemotherapeutic agent,
lysosome inhibitor, immunotherapeutic agent, or calpain inhibit are
present in the same pharmaceutical composition.
[0345] In some embodiments, the compound of Formula III, or its
pharmaceutically acceptable salt, and the chemotherapeutic agent,
lysosome inhibitor, immunotherapeutic agent, or calpain inhibit are
present in the same pharmaceutical composition.
[0346] In some embodiments, there is a synergistic efficacy of
compounds of Formula I and traditional chemotherapeutics CPT-11 and
5-FU.
[0347] In some embodiments, there may be a synergistic efficacy of
compounds of Formula Ia and traditional chemotherapeutics CPT-11
and 5-FU.
[0348] In some embodiments, there may be a synergistic efficacy of
compounds of Formula II and traditional chemotherapeutics CPT-11
and 5-FU.
[0349] In some embodiments, there may be a synergistic efficacy of
compounds of Formula III and traditional chemotherapeutics CPT-11
and 5-FU.
[0350] The methods described herein, in addition to treating
cancer, can also reduce cancer growth and/or progression, inhibit
tumor growth, or prevent the spread or metastasis of cancer in a
mammal comprising administering to the mammal in need thereof a
therapeutically effective amount of a compound described herein or
pharmaceutically acceptable salt thereof, or a pharmaceutical
composition comprising the compound described herein or
pharmaceutically acceptable salt thereof. In some embodiments, one
or more compounds may be combined in the same composition for any
of the methods disclosed herein.
[0351] The present disclosure also provides methods for killing or
inhibiting growth of a cancer cell comprising contacting the cancer
cell with an effective amount of a compound or pharmaceutically
acceptable salt thereof, or a pharmaceutical composition comprising
the compound or salt.
[0352] Thus, the compounds can be used as anti-cancer and
anti-tumor agents, e.g., the compounds can kill or inhibit the
growth of cancer cells. The compounds can also be used in methods
of reducing cancer in an animal, or in methods of treating or
preventing the spread or metastasis of cancer in an animal, or in
methods of treating an animal afflicted with cancer. The compounds
can also be used in methods of killing or inhibiting the growth of
a cancer cell, or in methods of inhibiting tumor growth.
[0353] The compounds can be tested for anti-cancer activity by
methods known to those of skill in the art. Examples of anti-cancer
assays include, but are not limited to, standard cell viability
assays, such as the XTT assay, or by metabolic activity assays.
[0354] Generally, cancer refers to any malignant growth or tumor
caused by abnormal and uncontrolled cell division; it may spread to
other parts of the body through the lymphatic system or the blood
stream. Cancers include both solid tumors and blood-borne
tumors.
[0355] Cancers that are treatable are broadly divided into the
categories of carcinoma, lymphoma and sarcoma. Examples of
carcinomas include, but are not limited to: adenocarcinoma, acinic
cell adenocarcinoma, adrenal cortical carcinomas, alveoli cell
carcinoma, anaplastic carcinoma, basaloid carcinoma, basal cell
carcinoma, bronchiolar carcinoma, bronchogenic carcinoma,
renaladinol carcinoma, embryonal carcinoma, anometroid carcinoma,
fibrolamolar liver cell carcinoma, follicular carcinomas, giant
cell carcinomas, hepatocellular carcinoma, intraepidermal
carcinoma, intraepithelial carcinoma, leptomanigio carcinoma,
medullary carcinoma, melanotic carcinoma, menigual carcinoma,
mesometonephric carcinoma, oat cell carcinoma, squamal cell
carcinoma, sweat gland carcinoma, transitional cell carcinoma, and
tubular cell carcinoma. Sarcomas include, but are not limited to:
amelioblastic sarcoma, angiolithic sarcoma, botryoid sarcoma,
endometrial stroma sarcoma, ewing sarcoma, fascicular sarcoma,
giant cell sarcoma, granulositic sarcoma, immunoblastic sarcoma,
juxaccordial osteogenic sarcoma, coppices sarcoma, leukocytic
sarcoma (leukemia), lymphatic sarcoma (lympho sarcoma), medullary
sarcoma, myeloid sarcoma (granulocitic sarcoma), austiogenci
sarcoma, periosteal sarcoma, reticulum cell sarcoma (histiocytic
lymphoma), round cell sarcoma, spindle cell sarcoma, synovial
sarcoma, and telangiectatic audiogenic sarcoma. Lymphomas include,
but are not limited to: Hodgkin's disease and lymphocytic
lymphomas, such as Burkitt's lymphoma, NPDL, NML, NH and diffuse
lymphomas.
[0356] Thus, examples of cancers that can be treated using the
compounds described herein include, but are not limited to,
Hodgkin's disease, non-Hodgkin's lymphomas, acute lymphocytic
leukemia, multiple myeloma, breast carcinomas, ovarian carcinomas,
lung carcinomas, Wilms' tumor, testicular carcinomas, soft-tissue
sarcomas, chronic lymphocytic leukemia, primary macroglobulinemia,
bladder carcinomas, chronic granulocytic leukemia, primary brain
carcinomas, malignant melanoma, small-cell lung carcinomas, stomach
carcinomas, colon carcinomas, malignant pancreatic insulinoma,
malignant carcinoid carcinomas, malignant melanomas,
choriocarcinomas, mycosis fungoides, head and neck carcinomas,
osteogenic sarcoma, pancreatic carcinomas, acute granulocytic
leukemia, hairy cell leukemia, rhabdomyosarcoma, Kaposi's sarcoma,
genitourinary carcinomas, thyroid carcinomas, esophageal
carcinomas, malignant hypercalcemia, renal cell carcinomas,
endometrial carcinomas, polycythemia vera, essential
thrombocytosis, adrenal cortex carcinomas, skin cancer, and
prostatic carcinomas.
[0357] In some embodiments, the cancer is lung cancer (such as
non-small cell lung cancer), breast cancer, prostate cancer,
ovarian cancer, testicular cancer, colon cancer, renal cancer,
bladder cancer, pancreatic cancer, glioblastoma, neuroblastoma,
sarcomas such as Kaposi's sarcoma and Ewing's sarcoma, hemangiomas,
solid tumors, blood-borne tumors, rhabdomyosarcoma, CNS cancer
(such as brain cancer), retinoblastoma, neuroblastoma, leukemia,
melanoma, kidney or renal cancer, and osteosarcoma.
[0358] The compounds can be used in methods of killing or
inhibiting the growth of cancer cells, either in vivo or in vitro,
or inhibiting the growth of a cancerous tumor.
[0359] The present disclosure also provides pharmaceutical packs or
kits comprising one or more containers filled with one or more
compounds of Formula I described herein. The present disclosure
also provides pharmaceutical packs or kits comprising one or more
containers filled with one or more compounds of Formula Ia
described herein. The present disclosure also provides
pharmaceutical packs or kits comprising one or more containers
filled with one or more compounds of Formula II described herein.
The present disclosure also provides pharmaceutical packs or kits
comprising one or more containers filled with one or more compounds
of Formula III described herein.
[0360] Optionally associated with such container(s) can be a notice
in the form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals or biological products,
which notice reflects approval by the agency of manufacture, use or
sale for human administration for treating a condition, disease, or
disorder described herein. In some embodiments, the kit contains
more than one compound described herein. In some embodiments, the
kit comprises a compound described herein in a single injectable
dosage form, such as a single dose within an injectable device such
as a syringe with a needle.
[0361] The kits may further comprise a chemotherapeutic agent, a
lysosome inhibitor, an immunotherapeutic agent, or calpain
inhibitor.
[0362] In order that the subject matter disclosed herein may be
more efficiently understood, examples are provided below. It should
be understood that these examples are for illustrative purposes
only and are not to be construed as limiting the claimed subject
matter in any manner.
EXAMPLES
Example 1: Cell-Based Drug Screening for p53 Pathway-Restoring
Small Molecules
Materials and Methods
[0363] High-throughput screening was performed using a non-invasive
bioluminescence imaging in human colorectal cancer cell lines that
stably express a p53-regulated luciferase reporter. Cells were
seeded in 96-well plates (Greiner Bio-One) at a density of
1.times.10.sup.4 cells per well. p53 transcriptional activity was
imaged using an IVIS imaging system for a period of 3-4 hours.
Cell Lines and Culture Conditions
[0364] DLD-1, SW480, HCT116, and HCT116 p53' colorectal cancer cell
lines that stably express a p53-regulated luciferase reporter were
previously generated in our laboratory. RXF393 renal cancer cell
lines, and W138 and MRC5 normal lung cell fibroblasts were
purchased from ATCC. Cell lines were maintained in HyClone.TM.
Dulbecco's High Glucose Modified Eagles Medium (DMEM, GE
Healthcare), HyClone.TM. McCoy's 5A (GE Healthcare), HyClone.TM.
RPMI 1640 (GE Healthcare), or Eagle's Minimum Essential Medium
(EMEM, ATCC) containing 10% fetal bovine serum and 1%
penicillin/streptomycin (complete media) at 37.degree. C. in 5%
CO.sub.2, as recommended by ATCC.
CellTiter-Glo.RTM. Luminescent Cell Viability Assay
[0365] Cells were seeded in 96-well plates at a density of
5.times.10.sup.3 cells per well. 20 .mu.L of CellTiter-Glo.RTM.
Reagent was added directly to the wells, following the
manufacturer's protocol, and bioluminescence signal was determined
using an IVIS imaging system.
Knockdown Expression of p73, DR5, ATG5, and FADD Using siRNA
[0366] A total of 1.times.10.sup.5 cells/well were plated per well
in a 12-well plate in medium with 10% FBS without antibiotic.
Forward transfection of p73 siRNA (s14319, Ambion.RTM.), DR5
(sc-40237, Santa Cruz Biotechnology), atg5 (137766, Ambion.RTM.),
FADD (S16706, Ambion.RTM.) was performed using the
Lipofectamine.RTM. RNAiMAX Transfection Reagent (Life Technologies)
and incubated for 48 hours before treatment.
Overexpression of p53 R175H Mutant by Lentivirus Infection
[0367] HCT116 p53.sup.-/- cells (previously obtained from the
Vogelstein Laboratory, Johns Hopkins University) were infected with
a lentivirus vector containing the p53 R175H mutant
(pLenti6/V5-p53_R175H, Addgene). Cells were selected with
blasticidin (8 .mu.g/mL) containing media cultured for 10 days.
Blasticidin-resistant clones (pooled clones) were screened for
expression of the p53 R175H mutation by Western blot analysis with
p53 DO-1 antibody.
Knockdown Expression of NOXA by Lentivirus Infection
[0368] A NOXA shRNA plasmid construct was amplified according to
the manufacturer's recommendation (TRC Lentiviral Human PMAIP1
shRNA, Dharmacon). Plasmid DNA was isolated using the PureLink.RTM.
HiPure Plasmid Filter Maxiprep Kit (Invitrogen) according to the
manufacturer's instructions. Lentivirus production was performed by
transfecting HEK293T cells at a density of 8.times.10.sup.6 cells
per 10 cm dish with 1.6 .mu.g pMD2.G envelope plasmid, 3.2 .mu.g
psPAX2 packaging vector, 3.2 .mu.g plasmid DNA, and 24 .mu.L of
Lipofectamine.RTM. Transfection reagent 2000 (Life Technologies) in
a total volume of reaction of 1 mL of antibiotic free DMEM media
for a period of 6-10 hours. Media was then replaced with antibiotic
free DMEM. Lentiviral particles were collected between 48-72 hours.
SW480 cells (2.3.times.10.sup.6 cells per well in a 12-well plate)
were infected 1:1 (virus containing media: antibiotic free DMEM
media, total volume 1 mL) for a period of 24 hours. Then, media was
replaced with DMEM complete media for an additional 24 hours. At
this point, cells were split and seeded (20% confluent) for
selection in a 10 cm dish with puromycin (2.5 .mu.g/mL)-containing
complete DMEM media and cultured for 10 days. Puromycin containing
complete media was replaced every 2-3 days. Puromycin-resistant
clones were screened for knockdown of NOXA by Western blot analysis
with NOXA antibody.
Colony Formation Assay
[0369] Cells were seeded in 6-well plates at a density of 500 cells
per well. Cells were treated with CB002 small molecule for 24
hours. Then, cells were cultured in drug-free complete media for 15
days. During the course of 15 days, the media was changed every 2-3
days. At the end of the two weeks, media was removed, wells were
washed twice with Dulbecco's phosphate buffered saline (PBS) and
the colonies were fixed and stained with 10% methanol and 0.25%
crystal violet (Sigma-Aldrich) for 30 minutes. Wells were then
carefully washed with distilled and deionized water and allowed to
dry.
Apoptosis Assay
[0370] Apoptotic cells were quantified by sub-G1 analysis. Cells
were seeded at a density of 2.5.times.10.sup.5 to 5.times.10.sup.5
in a 6-well plate and treated for 48-72 hours. After treatment,
adherent cells were trypsinized and collected along with floating
cells, washed with PBS and fixed in 70% ethanol. Cells were then
incubated in a Phosphate-citric acid buffer (0.2 M
Na.sub.2HPO.sub.4+0.1 M Citric Acid, pH 7.8) at room temperature
for 5 minutes, spun down and resuspended for staining with 50
.mu.g/mL propidium iodide (PI) in the presence of 250 .mu.g/mL
pancreatic ribonuclease (RNase A). Sub-G1 analyses were performed
using an Epics Elite Epics flow cytometer (Coulter-Beckman).
Immunoblotting
[0371] After treatment, cells were harvested by trypsinization,
washed with PBS, and lysed with RIPA buffer (Sigma-Aldrich) for 30
minutes to 1 hour at 4.degree. C. Protein lysates were spun down
and supernatant was collected. Protein quantification was performed
using a Pierce.TM. BCA Protein Assay Kit (Thermo Fisher
Scientific). 1.times.NuPAGE.RTM. LDS sample buffer (Thermo Fisher
Scientific) and 2-Mercaptoethanol as the reducing agent
(Sigma-Aldrich) were added to protein lysates, followed by boiling
for 15 minutes at 95.degree. C. Equal total protein amounts samples
were loaded into NuPAGE.TM. Novex.TM. 4-12% Bis-Tris Protein Gels
(1.5 mm, Thermo Fisher Scientific) and gel eletrophoresis was
performed with NuPAGE.TM. MES SDS Running Buffer. Proteins were
transferred onto an Immobilon-P membrane (PVDF, EMD Millipore)
using a Bio-Rad system with a 10% Tris-Glycine and 10% methanol
transfer buffer diluted in distilled and deionized water. After
transfer, membranes were blocked with 10% milk in TBST solution and
then incubated overnight with primary antibody, washed with TBST
and incubated with secondary antibody for 1 hour. Incubations were
performed in 5% milk in TBST solution. Signal was detected by using
a chemiluminescent detection kit, followed by autoradiography. The
following antibodies were used: p53 (DO-1, 1:1000, Santa Cruz), p73
(1:1000, Bethyl Laboratories), p21 and NOXA (1:250, EMD Millipore),
DR5, FADD, cleaved caspase 3, cleaved caspase 8, cleaved PARP, and
LC3B (1:1000, Cell Signaling), and -actin (1:10000, Sigma).
Statistical Analysis
[0372] Data are presented as means.+-.SEM (three biological
replicates). To assess the statistical significance of the
differences, Two-way ANOVA for two comparisons was performed, with
p<0.05 defined as statistically significant. Comparisons were
made against the DMSO vehicle control.
Bioluminescence Assay
[0373] Cell-based screening of p53 transcriptional activity for
small molecule CB002 was accomplished using noninvasive
bioluminescence imaging in human colorectal cancer cell lines
SW480, DLD-1, DLD-1 p73.sup.-/-, HCT116, and HCT116 p53.sup.-/-.
These cell lines stably express a p53 reporter, PG13-luc. Cells
were seeded in opaque 96-well culture at a density of 5.times.104
cells/well. The cells were treated with CB002 at ranging doses with
DMSO controls. Bioluminescence in cells was imaged for p53
transcriptional activity at 2 hours and 24 hours using IVIS imaging
system (Xenogen).
Cell Titer-Glo Luminescent Cell Viability Assay
[0374] Cell lines at a concentration of 4.times.10.sup.3 cells/well
were seeded out on an opaque 96-well plate and treated with CB002
and Analog 11 in ranging doses starting from 200 .mu.mol/L with
DMSO controls. At 72 hours after treatment, cells were mixed with
30 .mu.L Cell Titer-Glo reagent and after ten minutes of room
temperature incubation were imaged using IVIS imaging system
(Xenogen).
FACS Assay
[0375] Cells were seeded out at 1.times.10.sup.6 cells/well on six
well plates and treated with CB002 and Analog 11 at ranging doses
with DMSO controls. Cells were harvested after 72 hours of
treatment, all cells including floating cells were fixed with
ethanol and stained with Propidium Iodide and then analyzed using
Epics Elite flow cytometer to measure the DNA content of the
stained cells.
Western Immunoblot Analysis
[0376] Proteins were isolated using NP40 Lysis Buffer (20 mmol/L
Tris-HCl (pH 7.4), 150 mmol/L NaCl, 5 mmol/L EDTA, 50 mmol/L NaF, 1
mmol/L glycerophosphate, 5 mmol/L Na.sub.4P.sub.2O.sub.7, 0.5%
NP40, and complete protease inhibitor cocktail (Roche)) and
electrophoresed through 4-12% SDS-PAGE followed by semi-dry
transfer to PVDF membranes. The PVDF membranes were incubated with
different antibodies including p21 (OP64-100UG, EMD Millipore
(world wide web at
emdmillipore.com/US/en/product/Antip21WAF1-(Ab-1)-Mouse-mAb-(EA10),
EMD_BIO-OP64)), PUMA (12450S, Cell Signaling Technology, world wide
web at
cellsignal.com/products/primary-antibodies/puma-d30c10-rabbit-mab/1245-
0), DR5 (3696S, Cell Signaling Technology, world wide web at
cellsignal.com/products/primary-antibodies/dr5-antibody/3696?N=4294956287-
&Ntt=3696s&fromPage=plp&_requestid=541668), p53(sc-126,
Santa Cruz, world wide web at
scbt.com/scbt/fr/product/p53-antibody-do-1), and RAN (610341, BD
Transduction Laboratories, world wide web at
bdbiosciences.com/us/reagents/research/antibodies-buffers/cell-biologyrea-
gents/cell-biology-antibodies/purified-mouse-anti-ran-20ran/p/610341)
in blocking buffer at 4.degree. C. overnight. Bound antibody will
be detected using IRDye secondary antibodies (LI-COR Biosciences,)
in Odyssey blocking buffer for 1 hour then imaged using the ODYSSEY
infrared imaging system.
Example 2: CB002 Restores p53-Dependent Transcriptional Reporter
Activity
[0377] In order to identify small molecules that could restore the
p53 signaling pathway, 50,000 small molecules from the Chembridge
Library were screened using a firefly luciferase human p53 reporter
assay system. SW480 colorectal cancer cells that stably express the
human p53 reporter were treated with compounds at various
concentrations from 0-100 .mu.M for 2 and 24 hours. This initial
screen identified CB002 (ID 7745998, IUPAC name:
8-anilino-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione) as a small
molecule capable of activating the luciferase reporter in a
dose-dependent manner. To further validate the effects of CB002,
the screening was expanded by testing effects on p53-dependent
reporter activity in DLD-1, wild-type HCT116, and p53-null
colorectal cancer HCT116 cell lines. In all three cell lines tested
the activity of the reporter was induced in a dose-dependent manner
at 3 and 24 hours (see, FIG. 1). Referring to FIG. 1,
representative images of luciferase-based p53-reporter activity
assays are shown at 3 hours (Panel A). Quantification of the
luciferase-based p53-reporter activity assays in three different
cell lines incubated from 3-24 hours with CB002 are shown (Panels
B-D). p53-reporter activity assays are shown at 2 hours for HCT116
p53-null cells (Panel E). Three replicates were performed for each
concentration of CB002 as indicated in the figure panels. These
results document that CB002 restores the p53-dependent
transcriptional activity of a reporter gene.
Example 3: A Favorable Therapeutic Index is Observed with CB002
Using Human Tumor and Normal Cell Lines
[0378] To begin establishing the potential of CB002 as a candidate
therapeutic agent, its therapeutic index was determined by treating
cancer (DLD-1, SW480, and RXF393) and normal (WI38 and MRC5) cell
lines with concentrations of CB002 ranging from 0-500 .mu.M for a
period of 72 hours and assessing cell viability by the
CellTiter-Glo.RTM. luminescence assay (see, FIG. 2). IC.sub.50
values were determined using GraphPad analyses and are listed in
Table 1. CB002 has a significant therapeutic index among the cells
tested. Normal cell lines have an IC.sub.50 value of approximately
650 .mu.M while in the panel of cancer cell lines tested IC.sub.50
values ranged from 96 .mu.M-400 .mu.M. SW480 was observed to be the
most sensitive cell line, followed by DLD-1, and RXF393 is the
least sensitive.
TABLE-US-00001 TABLE 1 CB002 IC.sub.50 values determined using
GraphPad analyses for the cell lines tested in FIG. 2 Cell Line
IC.sub.50(.mu.M) 95% CI R.sup.2 SW480 96.47 82.92-96.52 0.991 DLD-1
161.1 135.8-190.0 0.946 DLD-1 p73kd 239.7 230.2-249.6 0.992 RXF393
399.5 259.4-615.5 0.928 MRC5 647.0 578.3-723.8 0.906 WI38 641.7
606.6-678.9 0.921
Example 4: CB002 Qualitatively Decreases Colony Formation by
Colorectal Cancer Cell Lines
[0379] To further validate CB002 as a candidate therapeutic, its
capability to affect cell growth was determined by analyzing cancer
cell colony formation. Colony formation assays are widely used to
determine the ability of a single cell to proliferate and form a
colony in culture. This method offers the advantage of elucidating
the sensitivity of cells towards cytotoxic agents in a long-term
assay that may mimic the response seen in mouse models. CB002 was
observed to significantly decreases colony formation in SW480,
DLD-1, and p73 DLD-1 stable knockdown cells (see, FIG. 3).
Referring to FIG. 3, cells were treated with 50 .mu.M of CB002 for
a period of 24 hours, at which point media was replaced with
complete media for a period of 15 days. Images are from one of
three replicates. These results suggest that CB002 has cytotoxic
effects against human cancer cells in a long-term assay and that
the effects may not depend on p73.
Example 5: CB002 Induces Apoptotic Cell Death of Tumor Cells
[0380] To determine whether CB002 promotes cancer cell death
through apoptosis, SW480 cells were treated for 48 hours and
subjected to sub-G1 analyses. Referring to FIG. 4, a two-way ANOVA
statistical analysis was performed, with p<0.05 against DMSO
vehicle control. Three replicates were performed, and a
representative histogram is shown. SW480 DMSO vehicle control
sub-G1 population was 8.7% whereas treatment with 96 .mu.M CB002
showed a population of 32.6% (see, FIG. 4, right panels). This
significant increase in sub-G1 content upon CB002 treatment
indicates an augmentation in apoptotic cells. Furthermore, 96 .mu.M
CB002 treated W138 normal cells showed a sub-G1 population of 9.8%
as compared to the DMSO control where the sub-G1 content was 7.6%
(see, FIG. 4, left panels). The increase in Sub-G1 population in
WI38 cells was determined not to be statistically significant (see,
FIG. 4, bottom panel). Thus, CB002 induces cell death through
apoptosis specifically in cancer cell lines but not in normal
cells.
Example 6: CB002 Induces the Expression of Endogenous p53 Target
Genes in Mutant p53-Expressing Tumor Cells and Apoptotic Cell
Death
[0381] To further investigate the potential of CB002 as a p53
pathway-restoring compound, various cancer cell lines were treated
and probed for expression of endogenous p53 target genes, as well
as for markers of apoptotic cell death. SW480 cells were treated
with DMSO, 12, 25, and 50 .mu.M CB002 as concentrations below the
IC.sub.50 value. Referring to FIG. 5, whole cell lysates were
subjected to Western blot analysis. p73 was knocked-down by siRNA
in SW480 cells followed by CB002 treatment for 16 hours (Panel A).
DR5 was knocked-down by siRNA in DLD-1 cells followed by CB002
treatment for 16 hours (Panel B). NOXA was knocked-down by shRNA in
SW480 cells followed by CB002 treatment for 16 hours (Panel C).
Parental SW480 and SW480 NOXA stable knockdown cells treated with
CB002 for 48 hours were subjected to a sub-G1 analysis (Panels D
and E). A two-way ANOVA statistical analysis was performed for
results from Panel D, p<0.05 against DMSO vehicle control (Panel
E). Three replicates were performed, and a representative histogram
is shown. c-caspase 3 corresponds to the cleaved form of
full-length caspase-3.
[0382] As shown in FIG. 5, panel A, CB002 was found to increase the
expression of proteins involved in p53-dependent cell cycle arrest
and apoptosis, including p21 and DR5 (compare lane 1 to 2, 3, and
4). NOXA, a pro-apoptotic protein, was found to be increased by
CB002 concentrations below the IC.sub.50 (50 .mu.M) and at the
IC.sub.50 (96 .mu.M) in SW480 cells (see, FIG. 5, panel B, compare
lane 1 to 2, and 3). Altogether, these data indicate that the p53
pathway is activated by CB002 as demonstrated by the induction of
endogenous p53 target genes.
[0383] To validate that cell death was mediated through apoptosis,
PARP cleavage was further assessed. Upon CB002 treatment, cleaved
PARP expression was observed to increase upon CB002 treatment as
compared to vehicle control (see, FIG. 5, panel A, compare lane 1
to 2, 3, and 4). Taken together, these data indicate that CB002
induces the expression of p53 target genes and cell death in the
SW480 cell line. Similar results were observed in DLD-1 cells (data
not shown). To further investigate the mechanism of CB002 in
restoring the p53 pathway, p73 protein expression was efficiently
knocked down (see, FIG. 5, panel A, see lanes 5-8) and p53 target
gene expression was assessed.
[0384] CB002 treatment was found to increase the expression of p53
target genes p21 and DR5, and associated PARP cleavage in
p73-knockdown cells (see, FIG. 5, panel A, compare lane 5 to 6, 7,
and 8). Overall these data suggest that p73 may not play a critical
role in the mechanism of CB002 p53 pathway restoration or cell
death. In addition, CB002 treatment groups showed constant p53
protein expression levels as the DMSO control (see, FIG. 5, panel
A, compare lane 1 to 2, 3, and 4). This indicates that CB002 might
not have an effect on mutant p53 protein expression in SW480 cells.
Nonetheless, further experiments were performed to corroborate this
finding suggest some degradation effects towards certain p53
mutants (refer to FIG. 6 section).
Example 7: NOXA Protein is Required for CB002-Induced Cell Death of
Tumor Cells
[0385] To determine the role of p53 target genes DR5 and NOXA in
CB002-mediated cell death, DR5 was knocked down by siRNA and a
SW480 cell line stably transfected with NOXA shRNA plasmid
construct was generated. Adequate DR5 knockdown was achieved as
shown in FIG. 5, panel B (refer to lanes 4-6). DR5 knockdown in
SW480 cells treated with 50 and 96 .mu.M CB002 continued to induce
the expression of cleaved caspase 3 (c-caspase 3) and cleaved PARP
(see, FIG. 5, panel B, compare lane 4 to 5 and 6) as efficiently as
the scrambled siRNA-treated cells (see, FIG. 5, panel B, compare
lanes 2 and 3 to 5 and 6). Therefore, DR5 appears dispensable for
CB002-mediated cell death in the tumor cell lines that were
tested.
[0386] By contrast, when NOXA was efficiently knocked down (see,
FIG. 5, panel B, lanes 4-6), 50 and 96 .mu.M CB002 treatment did
not induce the expression of apoptotic markers c-caspase 3 and
cleaved PARP (see, FIG. 5, panel B, lanes 5-6). The requirement for
NOXA in tumor cell apoptosis induction after exposure to CB002 was
verified by conducting sub-G1 analyses. As expected, 96 .mu.M CB002
treatment in parental SW480 cells caused an increase in sub-G1
content (19%) as compared to DMSO treatment (4.5%). Nonetheless, 96
.mu.M CB002 treatment in SW480 where NOXA was stably knockeddown,
failed to increase the content of sub-G1 cells (3.7%) when compared
to DMSO treatment (3.93%) (see, FIG. 5, panels D and E). These data
denote NOXA as the primary mediator in the mechanism of action of
CB002-mediated cell death, in the tumor cell lines tested under the
described experimental conditions.
[0387] In addition to CB002 increasing the expression of apoptotic
markers, it was found to induce the expression of LC3B, a marker of
autophagy (see, FIG. 5, panel C, compare lane 1 to 2 and 3). NOXA
knock-down cells treated with 50 and 96 .mu.M CB002 failed to
induce LC3B expression (see, FIG. 5, panel C, lanes 5 and 6),
indicating that NOXA is required for autophagy induction. The role
of autophagy is further addressed below (see, FIG. 8).
Example 8: CB002 Treatment of Tumor Cells Destabilizes the R175H
p53 Mutant
[0388] As mutant p53 can acquire a gain-of-function activity,
targeting mutant p53 for degradation has been explored as a
therapeutic strategy. To investigate the capability of CB002 to
impact on mutant p53 protein expression, Western blot analysis was
used. CB002 treatments showed no alteration in p53 protein
expression levels with the DMSO control (see, FIG. 5, panel A,
compare lane 1 to 2, 3, and 4). To confirm these findings, a
cycloheximide chase assay was performed. Cells were treated with
vehicle control or CB002 for a period of 24 hours. Subsequently,
100 .mu.g/mL cycloheximide was added to the wells and protein
lysates were collected at different time points between 0-10 hours.
CB002 treatment did not affect mutant p53 stability in SW480 and
DLD-1 cells (see, FIG. 6, compare lanes 1-5 to 6-10). Referring to
FIG. 6, experiments with SW480 cells (Panels A and B) and analysis
of DLD-1 colorectal cancer cells (Panel C) are shown. Cells were
treated for a 24 hour period with DMSO, CB002 or positive control
followed by 100 .mu.g/mL of cycloheximide addition, and protein
stability was evaluated in a time course ranging from 0-10 hours.
Histone deacetylase inhibitor, SAHA and Hsp90 inhibitor
Geldanamycin (1 .mu.M GA) were used as positive controls.
[0389] CB002 treatment at 50 and 100 .mu.M in HCT116 p53-null cells
that exogenously expressed the R175H p53 mutant showed a decrease
in mutant p53 protein expression compared to the DMSO control at 16
and 24 hours (see, FIG. 7, panel A, compare lanes 7 to 8 and 9, and
10 to 11 and 12). To validate this result, a cycloheximide chase
experiment was performed, as shown in FIG. 7, panel B. Using 100
.mu.M CB002 treatment decreased the stability of mutant p53
compared to the vehicle control (see, FIG. 7, panel B, compare
lanes 1-4 to 5-8). To investigate that the data observed was not an
effect that is specific for exogenously expressed protein, RXF393
renal cancer cells that endogenously express R175H p53 were treated
with 100 and 150 .mu.M CB002. Both concentrations of CB002 at 16
and 24 hours were found to decrease R175H p53 mutant expression as
compared to the vehicle control (see, FIG. 7, panel C). Altogether,
the data suggests that CB002 is capable of decreasing mutant p53
expression but potentially in a mutation-selective manner. In
addition, CB002 p53 degradation was specific to mutant p53 as it
was unable to decrease the expression of wild-type p53 (see, FIG.
7, panel A, lanes 1-6).
[0390] Referring to FIG. 7, CB002 reduced the protein expression of
the exogenous R175H mutant in HCT116 p53-null cells and not the
HCT116 wild-type p53 cells (Panel A). HCT116 R175H p53 cells were
treated for 24 hours with DMSO or CB002 followed by 100 .mu.g/mL
cycloheximide addition. Protein stability was evaluated from 0-10
hours (Panel B). CB002 reduced the protein expression of the
endogenous R175H mutant p53 in RXF393 renal cancer cells (Panel C).
Co-treatment for 24 hours with proteasome inhibitor MG132 and CB002
rescued the expression of the R175H mutant p53, suggesting the
involvement of the ubiquitin proteasome system in CB002-mediated
mutant p53 degradation (Panel D).
Example 9: CR002-Dependent R175H Mutant p53 Degradation in Tumor
Cells is Rescued by MG132
[0391] Mutant p53 can be degraded by various mechanisms including
via MDM2-mediated degradation through the ubiquitin proteasome
system. In order to explore the route by which the R175H p53 mutant
was being degraded in response to CB002, HCT116 p53 R175H cells
were pretreated with the MDM2 inhibitor Nutlin-3A and the
proteasome inhibitor MG132 prior to CB002 treatment. After 1 hour
of incubation, cells were simultaneously treated with CB002 and
Nutlin-3A or MG132. Co-treatment using 50 or 100 .mu.M CB002 along
with Nutlin-3A for 24 hours still resulted in reduction of R175H
p53 mutant protein expression (see, FIG. 7, panel D, compare lane 4
to 5 and 6). These results suggest that MDM2 may not be required
for the decreased R175H mutant p53 stability in the presence of
CB002. On the other hand, treatment with CB002 and two different
concentrations of MG132 (5 and 10 .mu.M) appeared to rescue R175H
mutant p53 protein expression (see, FIG. 7, compare lanes 7 to 8
and 9, and 10 to 11 and 12). The data suggest that the R175H mutant
p53 protein is degraded in human cancer cells in response to CB002
in a manner that is dependent on the ubiquitin proteasome
system.
Example 10: Autophagy does not Play a Role in Mutant p53
Degradation but it Appears to be Required for Apoptotic Cell
Death
[0392] During CB002 treatment, cells formed vacuoles suggesting an
involvement of an autophagy process. In addition, because it has
been recently reported that mutant p53 can be degraded through the
lysosome, whether CB002 may induce autophagy as a potential
mechanism for R175H mutant p53 degradation was investigated. Using
an autophagy detection kit (CYTO-ID.RTM. autophagy detection kit,
Enzo.RTM. Life sciences) an increase in autophagic vacuole-specific
staining upon 50 .mu.M CB002 treatment compared to the DMSO control
in DLD-1 cells was observed (see, FIG. 8, panel A). To confirm
these findings, a Western blot was probed for accumulation of LC3B,
a well-known marker used in autophagy studies. Because accumulation
of LC3B can indicate a blockage of autophagy, LC3 conversion was
evaluated during a time course from 0-48 hours of treatment with
160 .mu.M CB002 in DLD-1 cells. FIG. 8, panel B shows that
following CB002 treatment there is an initial increase of LC3B and
not in the DMSO treatment (compare lane 1 (0 hr) to lane 2, and 8).
LC3B expression increases to a maximum in CB002 treated (see, FIG.
8, panel B, lane 10) followed by a drop of its protein expression.
No change in expression of LC3B was observed over time in DMSO
control treated cells (see, FIG. 8, Panel B, lanes 2-7). LC3B
initially increases in the process of autophagy, and later it is
degraded in the lysosomes. Thus, the data indicates that autophagy
is being induced by CB002 treatment.
[0393] To explore if the R175H mutant p53 was getting degraded
through the autophagy mechanism, autophagy was blocked using the
autophagosome/lysosome fusion inhibitor, chloroquine (Chl). As
expected, treatment with CB002 in HCT116 p53 R175H cells for 16 and
24 hours decreased mutant p53 expression (see, FIG. 8, Panel C,
compare lanes 1 to 2 and 3, and 7 to 8 and 9). Co-treatment of
CB002 and Chl for 16 and 24 hours did not rescue R175H mutant p53
protein expression, indicating that autophagy does not play a role
in CB002 mediated R175H mutant p53 degradation (see, FIG. 8, Panel
C, compare lanes 4 to 5 and 6, and 10 to 11 and 12).
[0394] As autophagy can be induced for cell survival during
cellular stress, its contribution to CB002-induced cell death was
investigated. Autophagy was blocked using Chl added to CB002 to
treat cells and the effects on apoptotic cell death markers were
evaluated. CB002 induced the expression of cleaved caspases and
PARP (see, FIG. 8, Panel D, lanes 2 and 3). Upon combination
treatment using CB002 and Chl, cleaved caspase 8 and cleaved PARP
were reduced compared to CB002 alone (see, FIG. 8, Panel D, compare
lanes 2 and 3 to 5 and 6). To further validate this result,
autophagy was down-regulated by efficient siRNA-mediated knockdown
of the autophagy related 5 (atg5) gene (see, FIG. 8, Panel E, lanes
4-6, and 10-12). Ablation of atg5 resulted in complete loss of PARP
cleavage upon treatment of CB002 for 16 and 24 hours (see, FIG. 8,
Panel E, lanes 5-6 and 11-12). Although autophagy is mostly thought
to be an adaptive process allowing the cell to survive during
stress, here it seems to be required for CB002 mediated apoptotic
cell death. Other investigators have demonstrated atg5 is
implicated in autophagic cell death induced by IFN-.gamma. via
interaction with Fas-associated protein with death domain (FADD), a
particular scenario that requires caspases. Since the data suggests
that atg5 is required for apoptosis, whether the atg5-FADD axis was
an interaction required for CB002 mediated cell death was explored.
As shown in FIG. 8, Panel F, lane 4-6, suitable knockdown of FADD
was achieved. CB002 treatment was able to induce the expression of
cleaved PARP in FADD knockdown cells (see, FIG. 8, Panel F, lane 5
and 6) as efficient as the scrambled siRNA (see, FIG. 8, Panel F,
lane 2 and 3). Thus, FADD is not crucial in CB002 mediated cell
death.
[0395] Referring to FIG. 8, CB002 induced autophagy as indicated by
specific recognition of autophagic vacuoles (detection using the
CYTO-ID autophagy detection kit at 24 hours) (Panel A) and LC3B
protein expression levels in DLD-1 cells (cells were treated with
160 .mu.M CB002 or DMSO control for a period of 2-48 hours (Panel
B). Autophagy inhibition by Chl does not rescue mutant p53 protein
expression in HCT116 R175H p53 (Panel C). Blocking the autophagy
process in SW480 cells with 50 .mu.M chloroquine (Chl) reduced the
expression of apoptotic markers cleaved caspase 8 (c-Caspase 8) and
cleaved PARP (24 hours) (Panel D). Inhibition of autophagy by Atg5
siRNA completely abolished PARP cleavage in SW480 cells (Panel E).
Atg5-FADD apoptosis axis was not involved in CB002 mediated
apoptosis in SW480 cells (24 hours) (Panel F).
Example 11: CB002 Restores p53 Pathway in Colorectal Cancer
Cells
[0396] To identify p53-restoring small molecules, 50000 small
molecular compounds of Chembridge library were screened using a
functional cell-based assay in SW480 cells which carry a p53-luc
reporter. One compound CB002 (ID is 7745998, IPUA name is
8-anilino-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione) was found
to increase p53 responsive bioluminescence (see, FIG. 9, Panels A,
B and C). A CB002-related compound (Analog 11; ID is 849102, IPUA
name is 8-anilino-1,3,7-trimethyl-3,7-dihydro-1Hpurine-2,6-dione)
in hembridge library (see, FIG. 9, Panel A) was identified. Similar
to CB002, Analog 11 increased p53 responsive bioluminescence in a
dose dependent manner in SW480 cells (see, FIG. 9, Panels B and
C).
[0397] Referring to FIG. 9, the molecular structure of CB002 and
Analog 11 (R1) are shown (Panel A). SW480 with p53 reporter cells
were treated with CB002 and Analog 11 in doses ranging from 0 to 20
.mu.mol/L (Panel B). p53 responsive bioluminescence was imagined by
IVIS. Data are representative of triplicate wells. The relative
increase of bioluminescence in panel B is shown (Panel C).
Colorectal cancer SW480, DLD-1, HCT116 and HCT116 p53-null cells
were treated with CB002 and Analog 11 for 2 and 24 hours (Panel D).
p53 responsive bioluminescence was imagined by IVIS. The relative
increased bioluminescence in panel D at 2 hours is shown (Panel E).
The relative increased bioluminescence in Panel D at 24 hours is
shown (Panel F).
[0398] CB002 and Analog 11 were further applied to four colorectal
cancer cell lines consisting of SW480, DLD1, HCT116, and HCT116 p53
null. As shown in FIG. 9, CB002 increased p53 responsive
bioluminescence in both SW480 (mutant p53 R273H, P309S) and DLD-1
(mutant p53 S241F) cells in a dose-dependent manner at 2 and 24
hours, as well as in HCT116 p53 null cells (see, FIG. 9, Panels D,
E and F).
[0399] For verification of bioluminescence data, Western blot
analysis of endogenous protein levels of p21, PUMA and DR5 (as
representative p53 targets) was conducted by varying drug dose and
a time course experiment. Referring to FIG. 10, DLD-1 and DLD-p73
knockdown cells (Panel A), HCT116 (Panel B), and HCT116 p53-null
cells (Panel C) were treated with CB002 and Analog 11 for 8 and 24
hours. Protein levels of p53 target genes were determined by
Western blot analysis. In DLD-1 cells (see, FIG. 10, Panel A), a
minor increase in p21 was observed at 8 hours in both CB002 and
Analog 11 at doses of 25 .mu.mol/L and 50 .mu.mol/L. At 24 hours
there was a moderate increase in p21 with the moderate dose of 12
.mu.mol/L of CB002 and 25 .mu.mol/L of Analog 11. A minor increase
in PUMA was observed at the 25 .mu.mol/L and 50 .mu.mol/L doses for
CB002, and an increase at the 25 .mu.mol/L but not the 50 .mu.mol/L
of Analog 11 at 8 hours. At 24 hours, an increase in PUMA was
observed with exposure to CB002 and Analog 11. DR5 was slightly
increased in cells treated with CB002 at 8 and 24 hours, but no
change of DR5 was observed in cells treated with Analog 11 in DLD-1
cells.
[0400] CB002 and Analog 11 were further applied to HCT116 p53-null
cells. As shown in FIG. 10, Panel C, in p53-null HCT116, p21
appeared to increase moderately with Analog 11 and CB002 at 8
hours. At 24 hours no consistent difference was observed for both
CB002 and Analog 11 at all dosages with an increase at the single
12 .mu.mol/L dose in Analog 11. PUMA appeared to increase at the 50
.mu.mol/L dose at 8 hours and in both the 25 .mu.mol/L and 50
.mu.mol/L doses of Analog 11. At 24 hours no consistent difference
in PUMA was observable except a slight decrease in the two Analog
11 dosages. On the basis of the data with p53 responsive
bioluminescence and p53 target gene expression, CB002 and Analog 11
restore p53 pathway responses in DLD-1 and HCT116 p53-null
cells.
[0401] The p53 pathway signaling in HCT116 cells which carry with
wild-type p53 was also examined. CB002 increased p53 responsive
bioluminescence at 2 hours (see, FIG. 9, Panels D, E and F).
Consistent with the data using bioluminescence, p21 appeared to
increase moderately in a dose dependent fashion for both CB002 and
Analog 11. PUMA expression appeared to increase only at 24 hours
with Analog 11 in HCT116 cells (see, FIG. 10, Panel B). These data
suggest that CB002 reactivates the p53 pathway in wild-type p53
expressing HCT116 cells.
Example 12: CB002 Decreases Mutant p53 Protein Levels in Colorectal
Cancer Cells
[0402] The effect of CB002 and Analog 11 on mutant p53 protein
levels was examined in cancer cells. Mutant p53 protein in SW480
cells and wild-type p53 in HCT116 cells were examined via Western
blot assay at 24 hours following treatment at varying doses with
CB002 and Analog 11. Referring to FIG. 11, SW480 and HCT116 cells
were treated with CB002 and Analog 11 for 24 hours. p53 protein was
determined by Western blotting using anti-p53 (DO-1). As shown in
FIG. 11, mutant p53 protein level appeared to decrease
significantly with increasing dose of CB002, and decrease
moderately with high dose Analog 11. In contrast, a moderate
increase in wild-type p53 was observed with increasing dose of
CB002 and Analog 11 in HCT116 cells.
Example 13: CB002 Restores p53 Pathway Signaling in Part Through
Activation of p73 in Mutant p53-Expressing Colorectal Cancer
Cells
[0403] CB002 treatment was applied to DLD-1 and p73 knockdown DLD-1
cells at varying doses. Western blot analysis was conducted using
DLD-1 and p73 knockdown DLD-1 at 8 hours and 24 hours post-exposure
to low ascending doses of CB002 and Analog 11. As shown in FIG. 10,
panel A, p21, PUMA and DR5 were increased in protein level at 8 and
24 hours in DLD-1 cells treated with CB002 and Analog 11. By
contrast, p21 expression was absent for both CB002 and Analog 11
compounds in p73 knock-down DLD-1 cells. PUMA appeared to have
increased expression at 8 hours for both compounds over control. At
24 hours, a reduction in PUMA was observed for Analog 11 with CB002
largely unchanged from control. DR5 appeared to be unchanged at 8
hours and at 24 hours with less protein level in p73 knockdown
DLD-1 cells compared with that in DLD-1 cells. p73 protein level
appeared to be unchanged in DLD-1 at 8 hours with a non-dose
dependent moderate increase at 24 hours over control. p73 protein
was not detected at both time points for both compounds in p73
knockdown DLD-1 cells. These results taken together suggest that
knockdown of p73 may have some impact on CB002 and Analog
11-restoring p53 pathway signaling in mutant p53-expressing cancer
cells.
Example 14: CB002 Induces Cell Death in Tumor Cells with No
Significant Effect on Normal Cells
[0404] Whether CB002 and Analog 11 repress cancer cell growth was
examined. To address this issue, cell viability and sub-G1 were
determined in colorectal cancer cell lines SW480, HCT116, HCT116
p53.sup.-/- upon treatment with CB002 and Analog 11. As shown in
FIG. 12 (Panels A, B, and C), CB002 and Analog 11 both reduced cell
viability in SW480, HCT116 and HT116 p53-null cells in a dose
dependent manner with CB002 having the greatest effect. Flow
cytometry was conducted to assess sub-G1 fraction in SW480, HCT116
and HCT116 p53-null cells at 72 hours post-treatment. Both CB002
and Analog 11 resulted in increased cells in sub-G1 fraction over
untreated controls in SW480, HCT116 and HCT116 p53-null cells in a
dose dependent manner. CB002 resulted in a greater percentage of
cells in sub-G1 fraction than Analog 11 in SW480 and HCT116 at a
dose of 200 .mu.M (see, FIG. 12, Panel D). These results suggest
that CB002 and Analog 11 induce cell death in colorectal cancer
cells. CB002 has higher anti-tumor efficacy as compared to Analog
11.
[0405] Referring to FIG. 12, cell viability of SW480 cells treated
with CB002 and Analog 11 at 72 hours is shown (Panel A). Cell
viability of HCT116 p53-null cells treated with CB002 and Analog 11
at 72 hours is shown (Panel B). Cell viability of HCT116 cells
treated with CB002 and Analog 11 at 72 hours is shown (Panel C).
Cell cycle profiles of cancer cells SW480, HCT116 and HCT116
p53-null cells are shown (Panel D). Cells were treated with CB002
and Analog 11 for 72 hours. Cell viability (Panels A, B, and C) was
normalized to DMSO as control. Data are expressed as
mean.+-.SD.
[0406] CB002 was applied to human normal fibroblast Wi38 cells.
IC.sub.50 of CB002 in Wi38 cells was much higher than those in
colorectal cancer cells, SW480, DLD-1, HCT116 and HCT116 p53-null
cells (see, FIG. 13, Panels A and B), suggesting that there is a
favorable therapeutic index between normal cells and cancer cells.
Flow cytometry showed relatively unchanged sub-G1 fraction in
normal Wi38 cells treated at the dose (200 .mu.M) of CB002 that
effectively increased 20% of cells in sub-G1 in SW480 cancer cells
at 72 hours (see, FIG. 13, Panel C).
[0407] Referring to FIG. 13, imaging of Cell Titer-Glo as a cell
viability assay of SW480 and Wi38 cells treated with CB002 and
Analog 11 for 72 hours is shown (Panel A). IC.sub.50 of CB002 in
cancer cells and normal fibroblast Wi38 cells based on the cell
viability is shown (Panel B). The cells were treated with CB002 for
72 hours. Cell cycle profiles of SW480 and Wi38 cells treated with
CB002 for 72 hours are shown (Panel C).
Example 15: CB002 Synergizes with CPT-11 and 5-FU to Suppress Cell
Growth in Colorectal Cancer
[0408] Given that 5-FU and CPT-11 are traditional chemotherapy for
colorectal cancer patients often used in combination treatment,
whether CB002 can synergize with CPT-11 or 5-FU in inducing cell
death in colorectal cancer cells was examined. To address this
question, combination treatment of CB002 with CPT-11 or 5-FU was
assessed with escalating doses in SW480 cells. Referring to FIG.
14, SW480 cells were treated with CB002 and CPT-11 or 5-FU for 72
hours. Imaging of CellTiter-Glo cell viability of SW480 treated
with CB002 and CPT-11 or 5-FU I shown (Panel A). Cell viability of
SW480 cells treated with CB002 and CPT-11 is shown (Panel B). Cell
viability of SW480 cells treated with CB002 and 5-FU is shown
(Panel C). Combination Index of CB002 and CPT-11 is shown (Panel
D). Combination Index of CB002 and 5-FU is shown (Panel E). Cell
viability was normalized to DMSO as control. *p<0.05.
Combination index (CI)<1, =1 and >1 indicate synergism,
additive effect, and antagonism in drug combination treatment.
[0409] As shown in FIG. 14, cell viability of cancer cells had
greater reduction with ascending doses of each agent with greater
toxicity observed in 5-FU over CPT-11 (see, FIG. 14, Panel A).
Combination treatment with CB002 (66 .mu.M) and CPT-11 (a series of
doses) significantly reduced cell viability in SW480 as compared to
the single agent treatments alone (see, FIG. 14, Panel B). Further
combination index (CI) analysis indicates a synergism of this
combination of CB002 and CPT-11 (see, FIG. 14, Panel D). Similar to
the combination treatment of CB002 and CPT-11, CB002 synergized
with 5-FU-induced cell death. As shown in FIG. 14, Panels C and E,
combinational treatment of CB002 (22 .mu.M and 66 .mu.M) with 5-FU
(60 .mu.g/ml) significantly reduced cell viability in SW480 cells
as compared with the single agent treatment alone. Combinational
index (CI<1.0) indicates a synergism of CB002 and 5-FU in cancer
cells.
Example 16: CB002 and Structural Analogs Activate the p53 Reporter
Activity in Colorectal Cancer Cells
[0410] Referring to FIG. 15, 2.times.10.sup.4 cells/well were
seeded in a 96-well plate. A serial dilution of 1-100 .mu.M of the
indicated CB002 analog was used to treat SW480 cells carrying the
luciferase p53 reporter. The data indicates that the luciferase p53
reporter activity is induced in a dose dependent manner by the
treatment of various structural analogs (6 hours).
[0411] Referring to FIG. 16, 1.times.10.sup.4 cells/well were
seeded in a 96-well plate. A serial dilution was performed to
determine the dose response for each indicated CB002 analog. The
IC.sub.50 for each CB002 analog was determined in SW480 cells by
the Cell Titer-Glo.RTM. Assay. Analog 4 was the most potent
compound.
Example 17: CB002 Promotes Mutant p53 Degradation which is Enhanced
by Ganetespib Treatment
[0412] Referring to FIG. 17, 2.5.times.10.sup.5 cells/well were
seeded in a 6-well plate and treated with either vehicle control or
25 .mu.M Analog 4 for a 48 hour treatment. Cells were then
subjected to propidium iodide staining cell cycle analysis. Sub-G1
content indicates that only SW480 cancer cells treated with analog
4 underwent apoptosis and not normal human fibroblast W138 cells.
Two-way ANOVA, **** p<0.0001.
[0413] Referring to FIG. 18, 5.times.10.sup.5 cells/well were
seeded in a 12 well plate. RXF393 cells were treated with 96 .mu.M
CB002 and/or 0.1 .mu.M Ganetesbip for 16 hours. p53 expression was
analyzed by Western Blot analysis and showed that Ganetespib
treatment enhanced CB002-mediated mutant p53 degradation in RXF393
cells.
[0414] Referring to FIG. 19 (panels A and B), 1.times.10.sup.4
cells/well of SW80 cells were seeded in a 96-well plate. The cells
were treated for 24 hours with a combination of 0-20 .mu.M
Ganetestib with 0-200 .mu.M CB002 (panel A), and 0-100 .mu.M
Irinotecan with 0-200 .mu.M Analog 11 (panel B). CompuSyn software
was used to analyze the combination index (CI) of both drugs. The
results suggest that CB002 synergizes with Ganetespib (see, panel
A) and Analog 11 synergizes with Irinotecan (see, panel B).
Example 18: CB002 and Derivatives Induce NOXA Protein Expression in
Colorectal and Multiple Myeloma Cancer Cells
[0415] Referring to FIG. 20 (panels A and B), 6.times.10.sup.5
cells/well were seeded in a 12-well plate. SW480 cells were treated
with either 12 or 25 .mu.M Analog 4 for 16 and 24 hours. The
expression of p53 targets DR5 and Noxa, p53 and apoptotic marker
cleaved Parp were analyzed by Western Blot analysis. Analog 4
appeared to restore the p53 pathway as indicated by increased
expression of DR5, Noxa, and cleaved Parp (see, panel A). Induction
of Noxa expression was a property shared by the 24 hour treatment
of 100 .mu.M CB002, 25 .mu.M Analog 4, and 100 .mu.M Analog 11 in
SW480 cells (see, panel B).
[0416] Referring to FIG. 20 (panel C), 1.times.10.sup.6 cells/well
were seeded in a 12-well plate. 8226 parental and resistant
multiple myeloma cell lines were treated with 100 .mu.M CB002, 25
.mu.M Analog 4, or 100 .mu.M Analog 11. Western Blot analysis
showed that Analog 4 and Analog 11 were able to induce Noxa
expression in 8226 bortezomib resistant cell line.
Example 19: CB002 Derivatives Sensitize Resistant Cells to
Bortezomib
[0417] Referring to FIG. 21 (panels A and B), 3.times.10.sup.4
cells/well of 8226 cells were seeded in a 96-well plate. The cells
were treated for 48 hours with a combination of 0-100 nM Bortezomib
with 0-200 .mu.M Analog 4 (see, panel A) and 0-100 nM Bortezomib
with 0-200 .mu.M Analog 11 (see, FIG. 21, panel B). CompuSyn
software was used to analyze the combination index of both drugs.
The results indicate that Analog 4 was able to synergyze the most
with Bortezomib when compared to Analog 11.
[0418] Referring to FIG. 21 (panel C), 3.times.10.sup.4 cells/well
of MM1S parental and Bortezomib MM1S resistant cells were seeded in
a 96-well plate. Cell viability was determined by the Cell
Titer-Glo.RTM. Assay. The results suggest that treating MM1S
Bortezomib resistant cells with 25 .mu.M Analog 4 is able to
sensitize MM1S resistant cells to Bortezomib treatment.
Example 20: CB002/Caffeine and Etoposide Combination have Different
Cell Cycle Effects
[0419] Referring to FIG. 22 (panels A-F), propidium iodide staining
cell cycle analysis for 24 hour treatment with DMSO (panel A),
CB002 (panel B), CB002 and etoposide (panel C), etoposide (panel
D), caffeine (panel E), and caffeine and etoposixe (panel F). These
reults demonstrate that caffeine can deregulate the cell cycle G2
checkpoint. On the contrary to caffeine, CB002 does not appear to
deregulate the G2 cell cycle checkpoint. The deregulation of the G2
checkpoint as a mechanism by caffeine prevents p53-expressing tumor
cells that are treated with DNA damaging drugs, such as etoposide,
from arresting to have time to repair, and thus sensitizes cancer
cells to cell death. As such, the mechanism of CB002 does not
involve deregulation of the G2 checkpoint.
Example 21: Noxa Induction is a Unique Property of CB002 and its
Analogs
[0420] Referring to FIG. 23 (panels A and B), DLD-1 cells (panel A)
and SW80 cells (panel B) were treated for 24 hours with the
indicated agents. Noxa protein expression was analyzed by Western
blot and showed that only the CB002 analogs were capable of
inducing Noxa expression, whereas caffeine, pentoxifylline, and
theophylline dis not induce Noxa expression.
[0421] Various modifications of the described subject matter, in
addition to those described herein, will be apparent to those
skilled in the art from the foregoing description. Such
modifications are also intended to fall within the scope of the
appended claims. Each reference (including, but not limited to,
journal articles, U.S. and non-U.S. patents, patent application
publications, international patent application publications, gene
bank accession numbers, and the like) cited in the present
application is incorporated herein by reference in its
entirety.
* * * * *