Note: Descriptions are shown in the official language in which they were submitted.
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CA 02524394 2008-11-13
NOVEL BENZODIAZEPINE COMPOUNDS,
COMPOSITIONS AND USES THEREOF
This PCT application claims priority to U.S. published Patent No.
2004/0220180, and
also U.S. issued Patent No. 7,276,348, which is a continuation in part of U.S.
published
Patent No. 2004/0176358, which is a continuation in part of U.S. published
Patent No.
2005/0261176.
FIELD OF THE INVENTION
The present invention relates to novel chemical compounds, methods for their
discovery, and their therapeutic use. In particular, the present invention
provides
benzodiazepine derivatives and related compounds and methods of using
benzodiazepine
derivatives and related compounds as therapeutic agents to treat a number of
conditions
associated with the faulty regulation of the processes of programmed cell
death,
autoimmunity, inflammation, hyperproliferation, and the like.
BACKGROUND OF THE INVENTION
Multicellular organisms exert precise control over cell number. A balance
between
cell proliferation and cell death achieves this homeostasis. Cell death occurs
in nearly every
type of vertebrate cell via necrosis or through a suicidal form of cell death,
known as
apoptosis. Apoptosis is triggered by a variety of extracellular and
intracellular signals that
engage a common, genetically programmed death mechanism.
Multicellular organisms use apoptosis to instruct damaged or -unnecessary
cells to
destroy themselves for the good of the organism. Control of the apoptotic
process therefore
is very important to normal development, for example, fetal development of
fingers and toes
requires the controlled removal, by apoptosis, of excess interconnecting
tissues, as does, the
formation of neural synapses within the brain. Similarly, controlled apoptosis
is responsible
for the sloughing off of the inner lining of the uterus (the endometrium) at
the start of
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menstruation. While apoptosis plays an important role in tissue sculpting and
normal
cellular maintenance, it is also the primary defense against cells and
invaders (e.g., viruses)
which threaten the well being of the organism.
Not surprisingly many diseases are associated with dysregulation of the
process of
cell death. Experimental models have established a cause-effect relationship
between
aberrant apoptotic regulation and the pathenogenicity of various neoplastic,
autoimmune
and viral diseases. For instance, in the cell mediated immune response,
effector cells (e.g.,
cytotoxic T lymphocytes "CTLs") destroy virus-infected cells by inducing the
infected cells
to undergo apoptosis. The organism subsequently relies on the apoptotic
process to destroy
the effector cells when they are no longer needed. Autoimmunity is normally
prevented by
the CTLs inducing apoptosis in each other and even in themselves. Defects in
this process
are associated with a variety of autoimmune diseases such as lupus
erythematosus and
rheumatoid arthritis.
Multicellular organisms also use apoptosis to instruct cells with damaged
nucleic
acids (e.g., DNA) to destroy themselves prior to becoming cancerous. Some
cancer-causing
viruses overcome this safeguard by reprogramming infected (transformed) cells
to abort the
normal apoptotic process. For example, several human papilloma viruses (HPVs)
have
been implicated in causing cervical cancer by suppressing the apoptotic
removal of
transformed cells by producing a protein (E6) which inactivates the p53
apoptosis promoter.
Similarly, the Epstein-Barr virus (EBV), the causative agent of mononucleosis
and Burkitt's
lymphoma, reprograms infected cells to produce proteins that prevent normal
apoptotic
removal of the aberrant cells thus allowing the cancerous cells to proliferate
and to spread
throughout the organism.
Still other viruses destructively manipulate a cell's apoptotic machinery
without
directly resulting in the development of a cancer. For example, the
destruction of the
immune system in individuals infected with the human immunodeficiency virus
(HIV) is
thought to progress through infected CD4+ T cells (about 1 in 100,000)
instructing
uninfected sister cells to undergo apoptosis.
Some cancers that arise by non-viral means have also developed mechanisms to
escape destruction by apoptosis. Melanoma cells, for instance, avoid apoptosis
by
inhibiting the expression of the gene encoding Apaf-1. Other cancer cells,
especially lung
and colon cancer cells, secrete high levels of soluble decoy molecules that
inhibit the
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initiation of CTL mediated clearance of aberrant cells. Faulty regulation of
the apoptotic
machinery has also been implicated in various degenerative conditions and
vascular
diseases.
It is apparent that the controlled regulation of the apoptotic process and its
cellular
machinery is vital to the survival of multicellular organisms. Typically, the
biochemical
changes that occur in a cell instructed to undergo apoptosis occur in an
orderly procession.
However, as shown above, flawed regulation of apoptosis can cause serious
deleterious
effects in the organism.
There have been various attempts to control and restore regulation of the
apoptotic
machinery in aberrant cells (e.g., cancer cells). For example, much work has
been done to
develop cytotoxic agents to destroy aberrant cells before they proliferate. As
such,
cytotoxic agents have widespread utility in both human and animal health and
represent the
first line of treatment for nearly all forms of cancer and hyperproliferative
autoimmune
disorders like lupus erythematosus and rheumatoid arthritis.
Many cytotoxic agents in clinical use exert their effect by damaging DNA
(e.g., cis-
diaminodichroplatanim(II) cross-links DNA, whereas bleomycin induces strand
cleavage).
The result of this nuclear damage, if recognized by cellular factors like the
p53 system, is to
initiate an apoptotic cascade leading to the death of the damaged cell.
However, existing cytotoxic chemotherapeutic agents have serious drawbacks.
For
example, many known cytotoxic agents show little discrimination between
healthy and
diseased cells. This lack of specificity often results in severe side effects
that can limit
efficacy and/or result in early mortality. Moreover, prolonged administration
of many
existing cytotoxic agents results in the expression of resistance genes (e.g.,
bcl-2 family or
multi-drug resistance (MDR) proteins) that render further dosing either less
effective or
useless. Some cytotoxic agents induce mutations into p53 and related proteins.
Based on
these considerations, ideal cytotoxic drugs should only kill diseased cells
and not be
susceptible to chemo-resistance.
One strategy to selectively kill diseased cells is to develop drugs that
selectively
recognize molecules expressed in diseased cells. Thus, effective cytotoxic
chemotherapeutic agents, would recognize disease indicative molecules and
induce (e.g.,
either directly or indirectly) the death of the diseased cell. Although
markers on some types
of cancer cells have been identified and targeted with therapeutic antibodies
and small
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molecules, unique traits for diagnostic and therapeutic exploitation are not
known for most
cancers. Moreover, for diseases like lupus, specific molecular targets for
drug development
have not been identified.
What are needed are improved compositions and methods for regulating the
apoptotic processes in subjects afflicted with diseases and conditions
characterized by faulty
regulation of these processes (e.g., viral infections, hyperproliferative
autoimmune
disorders, chronic inflammatory conditions, and cancers).
SUMMARY
The present invention provides novel compounds that find use in treating a
number
of diseases and conditions and that find use in research, compound screening,
and
diagnostic applications. The present invention also provides uses of these
novel
compounds, as well as the use of known compounds, that elicit particular
biological
responses (e.g., compounds that bind to particular target molecules and/or
cause particular
cellular events). Such compounds and uses are described throughout the present
application
and represent a diverse collection of compositions and applications.
Certain preferred compositions and uses are described below. The present
invention
is not limited to these particular compositions and uses.
The present invention provides a number of useful compositions as described
throughout the present application. Certain preferred embodiments of the
present involve
compositions include a composition comprising the following formula:
CH3
0
RI
CI
R2
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wherein Rl is selected from napthalalanine; phenol; 1-Napthalenol; 2-
Napthalenol;
Halogen - I
Halogen.
OCF3 and quinolines; wherein R2 is
selected from the group consisting of-
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6InrL n Inn, 't ruv rwvL
I I
I I
I
OH \ OH; CI ; N3 ; OH ;
Jwti
O C H3 ; and OH ; and wherein Rl and R2 include both R or S
enantiomeric forms and racemic mixtures. .
Other preferred embodiments of the present involve compositions include a
composition comprising the following formula:
R1 0
N
R4
R2
R3
R3 R3
R3
wherein Rl is selected from H, alkyl, or substituted alkyl; wherein R2 is
selected from
hydrogen, a hydroxy, an slkoxy, a halo, an amino, a lower-alkyl, a substituted
amino, an
acetylamino, a hydroxyamino, an aliphatic group having 1-8 carbons and 1-20
hydrogens, a
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substituted aliphatic group of similar size, a cycloaliphatic group consisting
of < 10 carbons,
a substituted cycloaliphatic group, an aryl, a heterocyclic; wherein R3 is
selected from H,
alkyl, or substituted alkyl, and wherein at most one substituent is a hydroxyl
subgroup;
01/ \ /
I
wherein R4 is selected from (CH2)nC(CH3)3; (CH2)nCH(CH3)2;
\I/
CH2(CH2)nCH3; dialkyl (all regioisomers);
difluoromethyl (all regioisomers); and wherein n
= 0 - 5; and wherein R1, R2, R3 and R4 include both R or S enantiomeric forms
and racemic
mixtures.
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Still other preferred embodiments of the present involve compositions include
a
composition comprising the following formula:
R, O
N
R4
iN
R2
R3
R3 R3
R3
wherein R1 is selected from H, alkyl, or substituted alkyl; wherein R2 is
selected from
hydrogen, a hydroxy, an alkoxy, a halo, an amino, a lower-alkyl, a substituted
amino, an
acetylamino, a hydroxyamino, an aliphatic group having 1-8 carbons and 1-20
hydrogens, a
substituted aliphatic group of similar size, a cycloaliphatic group consisting
of < 10 carbons,
a substituted cycloaliphatic group, an aryl, a heterocyclic; wherein R3 is
selected from H,
alkyl, or substituted alkyl, and wherein at most one substituent is a hydroxyl
subgroup;
I ( '
wherein R4 is selected from (CH2)nC(CH3)3; (CH2)nCH(CH3)2;
CH2(CH2)nCH3; dialkyl (all regioisomers)
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difluoromethyl (all regioisomers) . and wherein n
= 0 - 5; and wherein RI, R2, R3 and R4 include both R or S enantiomeric forms
and racemic
mixtures.
In other preferred embodiments, the present invention provides a
pharmaceutical
composition. In such embodiments, the present invention provides a compound
that binds
to oligomycin conferring protein, and an agent (e.g., resveratrol, picetannol,
estrogen,
lansoprazole).
The present invention also provides methods and compositions useful in
regulating
cellular death. In preferred embodiments, the present invention provides a
subject and a
composition comprising a formula selected from the group consisting of-
0 0
As
I
0
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(Ca2-
NH
O S
H
\
HNC H O
O
As OH
+H3N H` HO
C02
R
R R
R O
R
R
R 0 R R
R
OH
R R
R
R
OH
/ R
HO
R
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R
R R
R
R O
\ R
R
R R
R R
R 0
R R
R R
R R O
R R
R
OH
R H2C
R
N
R
R
R
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R O O R
H3CO \ \ / \ OCN3
HO R R OH
R R
wherein R is selected from hydrogen, a hydroxy, an alkoxy, a halo, an amino, a
lower-alkyl-
a substituted-amino, an acetylamino, a hydroxyamino, an aliphatic group having
1-8
carbons and 1-20 hydrogens, a substituted aliphatic group of similar size, a
cycloaliphatic
group consisting of < 10 carbons, a substituted cycloaliphatic group, an aryl,
and a
heterocyclic; and such a composition is administered to the subject.
In still other preferred embodiments, the present invention provides
compositions
and methods for regulating cellular proliferation. In such embodiments, the
present
invention provides a subject and a composition comprising a formula selected
from:
oss*
,o
As
I
0
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(CO2
NH
O S
H
HNe H O
O
As OH
+H3N HO
H C02
R
R R
R O
I
R
R
R 0 R R
R
OH
R R
R
R
OH
HO R
R
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R
R R
R
R O
R
/ R
R R
R R
R 0
R R
R O R R
R
R R
R
OH
R H2C
R
N
R R
R
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R O O R
H3CO \ ~ ~ \ OCH3
I I
HO R R OH
R R
wherein R is selected from hydrogen, a hydroxy, an alkoxy, a halo, an amino, a
lower-alkyl-
a substituted-amino, an acetylamino, a hydroxyamino, an aliphatic group having
1-8
carbons and 1-20 hydrogens, a substituted aliphatic group of similar size, a
cycloaliphatic
group consisting of < 10 carbons, a substituted cycloaliphatic group, an aryl,
and a
heterocyclic; and the compostion is administered to the subject.
The present invention provides a number of methods for influencing the fate of
cells,
tissues, and organisms. Certain preferred embodiments of the present involve
methods for
regulating cell death. In such embodiments, the present invention provides
target cells
having mitochondria and a composition comprising the following formula:
CH3
O
R,
N
CI
R2
wherein R1 comprises a hydrophobic aromatic group larger than benzene; wherein
R2
comprises a phenolic hydroxyl group; and wherein R1, and R2 include both R or
S
enantiomeric forms and racemic mixtures. In additional embodiments, the cells
are exposed
to the composition under conditions such that said composition binds to the
oligomycin
sensitivity conferring protein so as to increase superoxide levels or alter
cellular ATP levels
in said cells.
In other embodiments, target cells are in vitro cells. In other embodiments,
the
target cells are in vivo cells. In still other embodiments, the target cells
are ex vivo cells. In
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yet other embodiments, the target cells are cancer cells. In some embodiments,
the target
cells are selected from the group consisting of B cells, T cells, and
granulocytes.
In other embodiments used in the regulation of cellular death, the present
invention
also provides the following compositions:
CH3
O
R1
CI N
R2
wherein R1 is selected from group consisting of: napthalalanine; phenol; 1-
Napthalenol; 2-Napthalenol;
mss" ~
Halogen -
Halogen.
;
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OCF3 and
quinolines; wherein R2 is selected from the group consisting of:
I ~ I I
n J w rtirtirvL L
I I
OH OH; CI ; N3 ; OH ;
~ Lns n rwt,
OH ; and O C H3 ; and wherein Rl and R2 include both R or S
enantiomeric forms and racemic mixtures.
In preferred embodiments wherein the present invention regulates cellular
death,
exposure of the composition to target cells results in an increase in cell
death of the target
cells.
The present invention also provides methods and compositions for regulating
cellular proliferation. In such embodiments, the present invention provides
proliferating
target cells having mitochondria, and a composition comprising the following
formula:
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CH3
O
R1
N
CI
R2
wherein R1 comprises a hydrophobic aromatic group larger than benzene; wherein
R2
comprises a phenolic hydroxyl group; wherein Rl and R2 include both R or S
enantiomeric
forms and racemic mixtures; and wherein the cells are exposed to the
composition under
conditions such that the composition binds to the mitochondrial ATP synthase
complex so
as to increase superoxide levels or alter cellular ATP levels in the cells. In
preferred
embodiments, the composition binds to oligomycin sensitivity conferring
protein.
In some embodiments, the target cells are in vitro cells. In other
embodiments, the
target cells are in vivo cells. In still other embodiments, the target cells
are ex vivo cells. In
other embodiments, the target cells are cancer cells. In yet other
embodiments, the target
cells are selected from the group consisting of B cells, T cells, and
granulocytes. In still
further embodiments, the target cells are proliferating cells.
In other embodiments wherein the present invention regulates cellular
proliferation,
the present invention provides the following composition:
CH3
O
R,
CI N
R2
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wherein Rl is selected from napthalalanine; phenol; 1-Napthalenol; 2-
Napthalenol;
n a
Halogen - I
Halogen.
a a a
OCF3 and quinolines; wherein R2 is
selected from the group consisting of:
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I I I I
vwt, iw ti vwL
I I
OH OH; CI ; N3 ; OH ;
rJw1
w
OH ; and OCH3
Still other preferred embodiments of the present invention involve
compositions
comprising the following formula (including R and S enantiomers and racemic
mixtures):
R7
O
N
R6 R8
N
R,
R4
R2
R3
R5
wherein Rl, R2, R3 and R4 are selected from the group consisting of. hydrogen;
CH3; a
linear or branched, saturated or unsaturated aliphatic chain having at least 2
carbons; a
linear or branched, saturated or unsaturated aliphatic chain having at least 2
carbons, and
having at least one hydroxy subgroup; a linear or branched, saturated or
unsaturated
aliphatic chain having at least 2 carbons, and having at least one thiol
subgroup; a linear or
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branched, saturated or unsaturated aliphatic chain having at least 2 carbons,
wherein said
aliphatic chain terminates with an aldehyde subgroup; a linear or branched,
saturated or
unsaturated aliphatic chain having at least 2 carbons, and having at least one
ketone
subgroup; a linear or branched, saturated or unsaturated aliphatic chain
having at least 2
carbons; wherein said aliphatic chain terminates with a carboxylic acid
subgroup; a linear or
branched, saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at
least one amide subgroup; a linear or branched, saturated or unsaturated
aliphatic chain
having at least 2 carbons, and having at least one acyl group; a linear or
branched, saturated
or unsaturated aliphatic chain having at least 2 carbons, and having at least
one nitrogen
containing moiety (e.g.,nitro, nitrile, etc.); a linear or branched, saturated
or unsaturated
aliphatic chain having at least 2 carbons, and having at least one amine
subgroup; a linear or
branched, saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at
least one ether subgroup; a linear or branched, saturated or unsaturated
aliphatic chain
having at least 2 carbons, and having at least one halogen subgroup; a linear
or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons, and having
at least one
nitronium subgroup; wherein R5 is selected from the group consisting of. OH;
N02; NR';
OR'; wherein R' is selected from the group consisting of. a linear or
branched, saturated or
unsaturated aliphatic chain having at least one carbon; a linear or branched,
saturated or
unsaturated aliphatic chain having at least 2 carbons, and having at least one
hydroxyl
subgroup; a linear or branched, saturated or unsaturated aliphatic chain
having at least 2
carbons, and having at least one thiol subgroup; a linear or branched,
saturated or
unsaturated aliphatic chain having at least 2 carbons, wherein said aliphatic
chain terminates
with an aldehyde subgroup; a linear or branched, saturated or unsaturated
aliphatic chain
having at least 2 carbons, and having at least one ketone subgroup; a linear
or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons; wherein
said aliphatic
chain terminates with a carboxylic acid subgroup; a linear or branched,
saturated or
unsaturated aliphatic chain having at least 2 carbons, and having at least one
amide
subgroup; a linear or branched, saturated or unsaturated aliphatic chain
having at least 2
carbons, and having at least one acyl group; a linear or branched, saturated
or unsaturated
aliphatic chain having at least 2 carbons, and having at least one nitrogen
containing moiety
(e.g., nitro, nitrile, etc.); a linear or branched, saturated or unsaturated
aliphatic chain having
at least 2 carbons, and having at least one amine subgroup; a linear or
branched, saturated or
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unsaturated aliphatic chain having at least 2 carbons, and having at least one
halogen
subgroup; a linear or branched, saturated or unsaturated aliphatic chain
having at least 2
carbons, and having at least one nitronium subgroup; wherein R6 is selected
from the group
consisting of. Hyrdrogen; NO2; Cl; F; Br; I; SR'; and NR'2, wherein R' is
defined as above
in R5; wherein R7 is selected from the group consisting of. Hydrogen; a linear
or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons; and
wherein R8 is an
aliphatic cyclic group larger than benzene; wherein said larger than benzene
comprises any
chemical group containing 7 or more non-hydrogen atoms, and is an aryl or
aliphatic cyclic
group. In some embodiments, R' is any functional group that protects the
oxygen of R5
from metabolism in vivo, until the compound reaches its biological target
(e.g.,
mitochondria). In some embodiments, R' protecting group(s) is metabolized at
the target
site, converting R5 to a hydroxyl group.
Additionally, in preferred embodiments R5 functions in interacting with
cellular
mitochondria (i.e., in the absence of R5, the compound has reduced binding
affinity for a
mitochondrial component). In further embodiments, Rl-R4 function to prevent
undesired
metabolism of the composition, and in particular a hydroxyl group at R5. In
yet other
embodiments, Rl-R4 function to promote cellular mitochondrial metabolism of
the
composition. In other preferred embodiments, the interacting of the
composition with
cellular mitochondria comprises binding the OSCP. In even further embodiments,
the
binding of the OSCP causes an increase in superoxide levels. In other
preferred
embodiments, R5 functions in regulating cellular proliferation and regulation
cellular
apoptosis.
The present invention also provides compositions and methods for treating
compromised vessels. For example, the present invention provides compositions
and
methods for treating compromised cardiac vessels. In preferred embodiments,
the
compromised vessel is an occluded vessel. In some embodiments, the present
invention
provides a method of treating a compromised vessel, comprising the providing
of drug-
eluting stent media. In preferred embodiments, the drug-eluting stent media
comprises a
pharmaceutical composition of the present invention. In preferred embodiments,
the
pharmaceutical composition is coated onto the drug-eluting stent media. In
further
embodiments, the pharmaceutical composition comprises an agent and a
pharmaceutically
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acceptable excipient. In preferred embodiments, the agent comprises any of the
structures
described herein.
Within the compositions and methods for treating compromised vessels in a
subject
suffering from a compromised vessel, the present invention further involves
treating said
subject with drug-eluting stent media and applying the pharmaceutical
composition onto the
compromised vessel. In some embodiments, the application of the pharmaceutical
composition onto said compromised vessel inhibits restenosis. In yet further
embodiments,
the application of the pharmaceutical composition inhibits smooth muscle cell
differentiation, migration and proliferation.
In other embodiments, the pharmaceutical composition further comprises an
adhesive agent. In some embodiments, the adhesive agent is biodegradable. In
even further
embodiments, the adhesive agent is fibrin glue. In certain embodiments, the
present
invention further provides a method of identifying therapeutic compositions.
In some
embodiments, the method provides a sample comprising mitochondrial F1Fo ATPase
and a
candidate F1Fo ATPase inhibitor. In further embodiments, the sample is
contacted with the
inhibitor. In further embodiments, the kcat/Km of said mitochondrial F1Fo-
ATPase is
measured, and the compositions that bind predominantly a F1Fo-ATPase-substrate
complex
and that do not alter said kcat/Km ratio of said mitochondrial F1Fo ATPase
upon binding of
said mitochondrial F1Fo-ATPase are selected as therapeutic compositions.
In some preferred embodiments, the method further comprises the step of
testing the
selected compositions in an animal to identify low toxicity and ability to
treat an
autoimmune disorder.
In other preferred embodiments, the sample further comprises mitochondria. In
other embodiments, the F1Fo ATPase is a pure enzyme. In even further
embodiments, the
F1Fo ATPase is located in a sub-mitochondrial particle.
In further preferred embodiments, the kcat/Km ratio is measured by determining
the
rate of ATP hydrolysis or synthesis as a function of ATP concentration. In
even further
embodiments, the kcat/Km ratio is calculated from Km Vmax, and km.
In further preferred embodiments, the selected compositions have high
inhibitory
activity at high substrate concentration and low activity at low substrate
concentration.
In certain embodiments, the present invention provides a method of treating
autoimmune disorders. In such embodiments, a subject and a composition capable
of
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binding mitochondrial F1Fo ATPase while not altering the F1Fo ATPase kcat/K,,,
ratio is
provided, and the composition is administered to the subject.
DESCRIPTION OF THE FIGURES
Figure 1 shows data demonstrating that the OSCP component is a binding protein
for Bz-423.
Figure 2 is a graph showing the binding isotherm of Bz-423 and purified human
OSCP.
Figure 3 shows siRNA regulation of OSCP.
Figure 4 shows data showing gene expression profiles of cells treated by the
compounds of the present invention. Data from an expression analysis for genes
up-
regulated in the presence of Bz-423 is presented in Figure 4A. Data from an
expression
analysis for genes down-regulated in the presence of Bz-423 is presented in
Figure 4B.
Data from an expression analysis for genes up-regulated in the presence of Bz-
OMe is
presented in Figure 4C. Data from an expression analysis for genes down-
regulated in the
presence of Bz-OMe is presented in Figure 4D.
DEFINITIONS
To facilitate an understanding of the present invention, a number of terms and
phrases are defined below.
As used herein, the term "benzodiazepine" refers to a seven membered non-
aromatic
heterocyclic ring fused to a phenyl ring wherein the seven-membered ring has
two nitrogen
atoms, as part of the heterocyclic ring. In some aspects, the two nitrogen
atoms are in 1 and
4 positions, as shown in the general structure below.
9 8 25
O5N
7 6 The benzodiazepine can be substituted with one keto group (typically at
the 2-
position), or with two keto groups, one each at the 2- and 5- positions. When
the
benzodiazepine has two keto groups, one each at the 2- and 5- positions, it is
referred to as
benzodiazepine-2,5-dione. Most generally, the benzodiazepine is further
substituted either
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on the six-membered phenyl ring or on the seven-membered heterocyclic ring or
on both
rings by a variety of substituents. These substituents are described more
fully herein.
The term "larger than benzene" refers to any chemical group containing 7 or
more
non-hydrogen atoms.
As used herein, the term "substituted aliphatic" refers to an alkane
possessing less
than 10 carbons where at least one of the aliphatic hydrogen atoms has been
replaced by a
halogen, an amino, a hydroxy, a nitro, a thio, a ketone, an aldehyde, an
ester, an amide, a
lower aliphatic, a substituted lower aliphatic, or a ring (aryl, substituted
aryl, cycloaliphatic,
or substituted cycloaliphatic, etc.). Examples of such include, but are not
limited to, 1-
chloroethyl and the like.
As used herein, the term "substituted aryl" refers to an aromatic ring or
fused
aromatic ring system consisting of no more than three fused rings at least one
of which is
aromatic, and where at least one of the hydrogen atoms on a ring carbon has
been replaced
by a halogen, an amino, a hydroxy, a nitro, a thio, a ketone, an aldehyde, an
ester, an amide,
a lower aliphatic, a substituted lower aliphatic, or a ring (aryl, substituted
aryl,
cycloaliphatic, or substituted cycloaliphatic). Examples of such include, but
are not limited
to, hydroxyphenyl and the like.
As used herein, the term "cycloaliphatic" refers to a cycloalkane possessing
less than
8 carbons or a fused ring system consisting of no more than three fused
cycloaliphatic rings.
Examples of such include, but are not limited to, decalin and the like.
As used herein, the term "substituted cycloaliphatic" refers to a cycloalkane
possessing less than 10 carbons or a fused ring system consisting of no more
than three
fused rings, and where at least one of the aliphatic hydrogen atoms has been
replaced by a
halogen, a nitro, a thio, an amino, a hydroxy, a ketone, an aldehyde, an
ester, an amide, a
lower aliphatic, a substituted lower aliphatic, or a ring (aryl, substituted
aryl, cycloaliphatic,
or substituted cycloaliphatic). Examples of such include, but are not limited
to, 1-
chlorodecalyl, bicyclo-heptanes, octanes, and nonanes (e.g., nonrbomyl) and
the like.
As used herein, the term "heterocyclic" refers to a cycloalkane and/or an aryl
ring
system, possessing less than 8 carbons, or a fused ring system consisting of
no more than
three fused rings, where at least one of the ring carbon atoms is replaced by
oxygen,
nitrogen or sulfur. Examples of such include, but are not limited to,
morpholino and the
like.
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As used herein, the term "substituted heterocyclic" refers to a cycloalkane
and/or an
aryl ring system, possessing less than 8 carbons, or a fused ring system
consisting of no
more than three fused rings, where at least one of the ring carbon atoms is
replaced by
oxygen, nitrogen or sulfur, and where at least one of the aliphatic hydrogen
atoms has been
replaced by a halogen, hydroxy, a thio, nitro, an amino, a ketone,\an
aldehyde, an ester, an
amide, a lower aliphatic, a substituted lower aliphatic, or a ring (aryl,
substituted aryl,
cycloaliphatic, or substituted cycloaliphatic). Examples of such include, but
are not limited
to 2-chloropyranyl.
As used herein, the term "linker" refers to a chain containing up to and
including
eight contiguous atoms connecting two different structural moieties where such
atoms are,
for example, carbon, nitrogen, oxygen, or sulfur. Ethylene glycol is one non-
limiting
example.
As used herein, the term "lower-alkyl-substituted-amino" refers to any alkyl
unit
containing up to and including eight carbon atoms where one of the aliphatic
hydrogen
atoms is replaced by an amino group. Examples of such include, but are not
limited to,
ethylamino and the like.
As used herein, the term "lower-alkyl-substituted-halogen" refers to any alkyl
chain
containing up to and including eight carbon atoms where one of the aliphatic
hydrogen
atoms is replaced by a halogen. Examples of such include, but are not limited
to, chlorethyl
and the like.
As used herein, the term "acetylamino" shall mean any primary or secondary
amino
that is acetylated. Examples of such include, but are not limited to,
acetamide and the like.
The term "derivative" of a compound, as used herein, refers to a chemically
modified compound wherein the chemical modification takes place either at a
functional
group of the compound or on the aromatic ring. Non-limiting examples of 1,4-
benzodiazepine derivatives of the present invention may include N-acetyl, N-
methyl, N-
hydroxy groups at any of the available nitrogens in the compound. Additional
derivatives
may include those having a trifluoromethyl group on the phenyl ring.
The term "stent" or "drug-eluting stent," as used herein, refers to any device
which
when placed into contact with a site in the wall of a lumen to be treated,
will also place
fibrin at the lumen wall and retain it at the lumen wall. This can include
especially devices
delivered percutaneously to treat coronary artery occlusions and to seal
dissections or
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aneurysms of splenic, carotid, iliac and popliteal vessels. The stent can also
have underlying
polymeric or metallic structural elements onto which the fibrin is applied or
the stent can be
a composite of fibrin intermixed with a polymer. For example, a deformable
metal wire
stent such as that disclosed in "U.S. Pat. No.: 4,886,062,
could be coated with fibrin as set forth above in one or more coats (i.e.,
polymerization of
fibrin on the metal framework by application of a fibrinogen solution and a
solution of a
fibrinogen-coagulating protein) or provided with an attached fibrin preform
such as an
encircling film of fibrin. The stent and fibrin could then be placed onto the
balloon at a
distal end of a balloon catheter and delivered by conventional percutaneous
means (e.g. as
in an angioplasty procedure) to the site of the restriction or closure to be
treated where it
would then be expanded into contact with the body lumen by inflating the
balloon. The
catheter can then be withdrawn, leaving the fibrin stent of the present
invention in place at
the treatment site. The stent may therefore provide both a supporting
structure for the lumen
at the site of treatment and also a structure supporting the secure placement
of fibrin at the
lumen wall. Generally, a drums eluting stent allows for an active release of a
particular drug
at the stent implementation site.
As used herein, the term "catheter" refers generally to a tube used for
gaining access
to a body cavity or blood vessel.
As used herein, the term "valve" or "vessel" refers to any lumen within a
mammal.
Examples include, but are not limited to, arteries, veins, capillaries, and
biological lumen.
As used herein, the term "restenosis" refers to any valve which is narrowed.
Examples include, but are not limited to, the reclosure of a peripheral or
coronary artery
following trauma to that artery caused by efforts to open a stenosed portion
of the artery,
such as, for example, by balloon dilation, ablation, atherectomy or laser
treatment of the
artery.
As used herein, "angioplasty" or "balloon therapy" or "balloon angioplasty" or
"percutaneous translumina] coronary angioplasty" refers to a method of
treating blood
vessel disorders that involves the use of a balloon catheter to enlarge the
blood vessel and
thereby improve blood flow.
As used herein, "cardiac catheterization" or "coronary angiogram" refers to a
test
used to diagnose coronary artery disease using a catheterization procedure.
Such a
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procedure may involve, for example, the injection of a contrast dye into the
coronary
arteries via a catheter, permitting the visualization of a narrowed or blocked
artery.
As used herein, the teen "subject" refers to organisms to be treated by the
methods
of the present invention. Such organisms preferably include, but are not
limited to,
mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines,
and the like),
and most preferably includes humans. In the context of the invention, the term
"subject"
generally refers to an individual who will receive or who has received
treatment (e.g.,
administration of benzodiazepine compound(s), and optionally one or more other
agents)
for a condition characterized by the dysregulation of apoptotic processes.
The term "diagnosed," as used herein, refers to the to recognition of a
disease by its
signs and symptoms (e.g., resistance to conventional therapies), or genetic
analysis,
pathological analysis, histological analysis, and the like.
As used herein, the terms "anticancer agent," or "conventional anticancer
agent"
refer to any chemotherapeutic compounds, radiation therapies, or surgical
interventions,
used in the treatment of cancer.
As used herein the term, "in vitro" refers to an artificial environment and to
processes or reactions that occur within an artificial environment. In vitro
environments
include, but are not limited to, test tubes and cell cultures. The term "in
vivo" refers to the
natural environment (e.g., an animal or a cell) and to processes or reaction
that occur within
a natural environment.
As used herein, the term "host cell" refers to any eukaryotic or prokaryotic
cell (e.g.,
mammalian cells, avian cells, amphibian cells, plant cells, fish cells, and
insect cells),
whether located in vitro or in vivo.
As used herein, the term "cell culture" refers to any in vitro culture of
cells. Included
within this term are continuous cell lines (e.g., with an immortal phenotype),
primary cell
cultures, finite cell lines (e.g., non-transformed cells), and any other cell
population
maintained in vitro, including oocytes and embryos.
In preferred embodiments, the "target cells" of the compositions and methods
of the
present invention include, refer to, but are not limited to, lymphoid cells or
cancer cells.
Lymphoid cells include B cells, T cells, and granulocytes. Granulocyctes
include
eosinophils and macrophages. In some embodiments, target cells are
continuously cultured
cells or uncultered cells obtained from patient biopsies.
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Cancer cells include tumor cells, neoplastic cells, malignant cells,
metastatic cells, and
hyperplastic cells. Neoplastic cells can be benign or malignant. Neoplastic
cells are benign if
they do not invade or metastasize. A malignant cell is one that is able to
invade and/or
metastasize. Hyperplasia is a pathologic accumulation of cells in a tissue or
organ, without
significant alteration in structure or function.
In one specific embodiment, the target cells exhibit pathological growth or
proliferation. As used herein, the term "pathologically proliferating or
growing cells" refers to
a localized population of proliferating cells in an animal that is not
governed by the usual
limitations of normal growth.
As used herein, the term "un-activated target cell" refers to a cell that is
either in the Go
phase or one in which a stimulus has not been applied.
As used herein, the term "activated target lymphoid cell" refers to a lymphoid
cell
that has been primed with an appropriate stimulus to cause a signal
transduction cascade, or
alternatively, a lymphoid cell that is not in G,, phase. Activated lymphoid
cells may
proliferate, undergo activation induced cell death, or produce one or more of
cytotoxins,
cytokines, and other related membrane-associated proteins characteristic of
the cell type
(e.g., CD8+ or CD4+). They are also capable of recognizing and binding any
target cell that
displays a particular antigen on its surface, and subsequently releasing its
effector
molecules.
As used herein, the term "activated cancer cell" refers to a cancer cell that
has been
primed with an appropriate stimulus to cause a signal transduction. An
activated cancer cell
may or may not be in the Go phase.
An activating agent is a stimulus that upon interaction with a target cell
results in a
signal transduction cascade. Examples of activating stimuli include, but are
not limited to,
small molecules, radiant energy, and molecules that bind to cell activation
cell surface
receptors. Responses induced by activation stimuli can be characterized by
changes in,
among others, intracellular Ca2+, superoxide, or hydroxyl radical levels; the
activity of
enzymes like kinases or phosphatases; or the energy state of the cell. For
cancer cells,
activating agents also include transforming oncogenes.
In one aspect, the activating agent is any agent that binds to a cell surface
activation
receptor. These can be selected from the group consisting of a T cell receptor
ligand, a B cell
activating factor (`GAFF"), a TNF, a Fas ligand (FasL), a CD40 ligand, a
proliferation
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inducing ligand ("APRIL"), a cytokine, a chemokine, a hormone, an amino acid
(e.g.,
glutamate), a steroid, a B cell receptor ligand, gamma irradiation, UV
irradiation, an agent or
condition that enhances cell stress, or an antibody that specifically
recognizes and binds a cell
surface activation receptor (e.g., anti-CD4, anti-CD8, anti-CD20, anti-TACI,
anti-BCMA, anti-
TNF receptor, anti-CD40, anti-CD3, anti-CD28, anti-B220, anti-CD38, and-CD19,
and anti-
CD21). BCMA is B cell maturation antigen receptor and TACI is transmembrane
activator and
CAML interactor.
Antibodies include monoclonal or polyclonal or a mixture thereof.
Examples of a T cell ligand include, but are not limited to, a peptide that
binds to an
MHC molecule, a peptide MHC complex, or an antibody that recognizes components
of the T
cell receptor.
Examples of a B cell ligand include, but are not limited to, a molecule or
antibody that
binds to or recognizes components of the B cell receptor.
Examples of reagents that bind to a cell surface activation receptor include,
but are not
limited to, the natural ligands of these receptors or antibodies raised
against them (e.g., anti-
CD20). RITUXIN (Genentech, Inc., San Francisco, CA) is a commercially
available anti-CD
chimeric monoclonal antibody.
Examples of agents or conditions that enhance cell stress include heat,
radiation,
oxidative stress, or growth factor withdrawal and the like. Examples of growth
factors include,
20 but are not limited to serum, IL-2, platelet derived growth factor
("PDGF"), and the like.
As used herein, the term "effective amount" refers to the amount of a compound
(e.g., benzodiazepine) sufficient to effect beneficial or desired results. An
effective amount
can be administered in one or more administrations, applications or dosages
and is not
limited intended to be limited to a particular formulation or administration
route.
As used herein, the term "dysregulation of the process of cell death" refers
to any
aberration in the ability of (e.g., predisposition) a cell to undergo cell
death via either
necrosis or apoptosis. Dysregulation of cell death is associated with or
induced by a variety
of conditions, including for example, autoimmune disorders (e.g., systemic
lupus
erythematosus, rheumatoid artluitis, graft-versus-host disease, myasthenia
gravis, Sjogren's
syndrome, etc.), chronic inflammatory conditions (e.g., psoriasis, asthma and
Crohn's
disease), hyperproliferative disorders (e.g., tumors, B cell lymphomas, T cell
lymphomas,
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etc.), viral infections (e.g., herpes, papilloma, HIV), and other conditions
such as
osteoarthritis and atherosclerosis.
It should be noted that when the dysregulation is induced by or associated
with a
viral infection, the viral infection may or may not be detectable at the time
dysregulation
occurs or is observed. That is, viral-induced dysregulation can occur even
after the
disappearance of symptoms of viral infection.
A "hyperproliferative disorder," as used herein refers to any condition in
which a
localized population of proliferating cells in an animal is not governed by
the usual
limitations of normal growth. Examples of hyperproliferative disorders include
tumors,
neoplasms, lymphomas and the like. A neoplasm is said to be benign if it does
not undergo,
invasion or metastasis and malignant if it does either of these. A metastatic
cell or tissue
means that the cell can invade and destroy neighboring body structures.
Hyperplasia is a
form of cell proliferation involving an increase in cell number in a tissue or
organ, without
significant alteration in structure or function. Metaplasia is a form of
controlled cell growth
in which one type of fully differentiated cell substitutes for another type of
differentiated
cell. Metaplasia can occur in epithelial or connective tissue cells. A typical
metaplasia
involves a somewhat disorderly metaplastic epithelium.
The pathological growth of activated lymphoid cells often results in an
autoimmune
disorder or a chronic inflammatory condition. As used herein, the term
"autoimmune
disorder" refers to any condition in which an organism produces antibodies or
immune cells
which recognize the organism's own molecules, cells or tissues. Non-limiting
examples of
autoimmune disorders include autoimmune hemolytic anemia, autoimmune
hepatitis,
Berger's disease or IgA nephropathy, Celiac Sprue, chronic fatigue syndrome,
Crohn's
disease, dermatomyositis, fibromyalgia, graft versus host disease, Grave's
disease,
Hashimoto's thyroiditis, idiopathic thrombocytopenia purpura, lichen planus,
multiple
sclerosis, myasthenia gravis, psoriasis, rheumatic fever, rheumatic arthritis,
scleroderma,
Sjorgren syndrome, systemic lupus erythematosus, type 1 diabetes, ulcerative
colitis,
vitiligo, and the like.
As used herein, the term "chronic inflammatory condition" refers to a
condition
wherein the organism's immune cells are activated. Such a condition is
characterized by a
persistent inflammatory response with pathologic sequelae. This state is
characterized by
infiltration of mononuclear cells, proliferation of fibroblasts and small
blood vessels,
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increased connective tissue, and tissue destruction. Examples of chronic
inflammatory
diseases include, but are not limited to, Crohn's disease, psoriasis, chronic
obstructive
pulmonary disease, inflammatory bowel disease, multiple sclerosis, and asthma.
Autoimmune diseases such as rheumatoid arthritis and systemic lupus
erythematosus can
also result in a chronic inflammatory state.
As used herein, the term "co-administration" refers to the administration of
at least
two agent(s) (e.g., benzodiazepines) or therapies to a subject. In some
embodiments, the co-
administration of two or more agents/therapies is concurrent. In other
embodiments, a first
agent/therapy is administered prior to a second agent/therapy. Those of skill
in the art
understand that the formulations and/or routes of administration of the
various
agents/therapies used may vary. The appropriate dosage for co-administration
can be
readily determined by one skilled in the art. In some embodiments, when
agents/therapies
are co-administered, the respective agents/therapies are administered at lower
dosages than
appropriate for their administration alone. Thus, co-administration is
especially desirable in
embodiments where the co-administration of the agents/therapies lowers the
requisite
dosage of a known potentially harmful (e.g., toxic) agent(s).
As used herein, the term "toxic" refers to any detrimental or harmful effects
on a cell
or tissue as compared to the same cell or tissue prior to the administration
of the toxicant.
As used herein, the term "pharmaceutical composition" refers to the
combination of
an active agent with a carrier, inert or active, making the composition
especially suitable for
diagnostic or therapeutic use in vivo, in vivo or ex vivo.
As used herein, the term "pharmaceutically acceptable carrier" refers to any
of the
standard pharmaceutical carriers, such as a phosphate buffered saline
solution, water,
emulsions (e.g., such as an oil/water or water/oil emulsions), and various
types of wetting
agents. The compositions also can include stabilizers and preservatives. For
examples of
carriers, stabilizers and adjuvants. (See e.g., Martin, Remington's
Pharmaceutical Sciences,
15th Ed., Mack Publ. Co., Easton, PA [1975]).
As used herein, the term "pharmaceutically acceptable salt" refers to any
pharmaceutically acceptable salt (e.g., acid or base) of a compound of the
present invention
which, upon administration to a subject, is capable of providing a compound of
this
invention or an active metabolite or residue thereof. As is known to those of
skill in the art,
"salts" of the compounds of the present invention may be derived from
inorganic or organic
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acids and bases. Examples of acids include, but are not limited to,
hydrochloric,
hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric,
glycolic, lactic,
salicylic, succinic, toluene-p-sulfonic, tartaric, acetic, citric,
methanesulfonic,
ethanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic,
benzenesulfonic acid, and
the like. Other acids, such as oxalic, while not in themselves
pharmaceutically acceptable,
may be employed in the preparation of salts useful as intermediates in
obtaining the
compounds of the invention and their pharmaceutically acceptable acid addition
salts.
Examples of bases include, but are not limited to, alkali metals (e.g.,
sodium)
hydroxides, alkaline earth metals (e.g., magnesium), hydroxides, ammonia, and
compounds
of formula NW4+, wherein W is C1_4 alkyl, and the like.
Examples of salts include, but are not limited to: acetate, adipate, alginate,
aspartate,
benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate,
camphorsulfonate,
cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,
fumarate,
flucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate,
hydrochloride,
hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,
methanesulfonate,
2-naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate, persulfate,
phenylpropionate, picrate, pivalate, propionate, succinate, tartrate,
thiocyanate, tosylate,
undecanoate, and the like. Other examples of salts include anions of the
compounds of the
present invention compounded with a suitable cation such as Na+, NH4+, and
NW4+
(wherein W is a C1.4 alkyl group), and the like.
For therapeutic use, salts of the compounds of the present invention are
contemplated as being pharmaceutically acceptable. However, salts of acids and
bases that
are non-pharmaceutically acceptable may also find use, for example, in the
preparation or
purification of a pharmaceutically acceptable compound.
As used herein, the terms "solid phase supports" or "solid supports," are used
in their
broadest sense to refer to a number of supports that are available and known
to those of
ordinary skill in the art. Solid phase supports include, but are not limited
to, silica gels,
resins, derivatized plastic films, glass beads, cotton, plastic beads, alumina
gels, and the
like. As used herein, "solid supports" also include synthetic antigen-
presenting matrices,
cells, liposomes, and the like. A suitable solid phase support may be selected
on the basis
of desired end use and suitability for various protocols. For example, for
peptide synthesis,
solid phase supports may refer to resins such as polystyrene (e.g., PAM-resin
obtained from
33
CA 02524394 2008-11-13
Bachem, Inc., Peninsula Laboratories, etc.), POLYHIPE) resin (obtained from
Aminotech,
Canada), polyamide resin (obtained from Peninsula Laboratories), polystyrene
resin grafted
with polyethylene glycol ('TENTAGELTM, Rapp Polymere, Tubingen, Germany) or
polydimethylacrylamide resin (obtained from MilligenBiosearch, California).
As used herein, the term "pathogen" refers a biological agent that causes a
disease
state (e.g., infection, cancer, etc.) in a host. "Pathogens" include, but are
not limited to,
viruses, bacteria, archaea, fungi, protozoans, mycoplasma, prions, and
parasitic organisms.
The terms "bacteria" and "bacterium" refer to all prokaryotic organisms,
including
those within all of the phyla in the Kingdom Procaryotae. It is intended that
the term
encompass all microorganisms considered to be bacteria including Mycoplasma,
Chlamydia, Actinomyces, Streptomyces, and Rickettsia. All forms of bacteria
are included
within this definition including cocci, bacilli, spirochetes, spheroplasts,
protoplasts, etc.
Also included within this term are prokaryotic organisms which are gram
negative or gram
positive. "Gram negative" and "gram positive" refer to staining patterns with
the
Gram-staining process which is well known in the art. (See e.g., Finegold and
Martin,
Diagnostic Microbiology, 6th Ed., CV Mosby St. Louis, pp. 13-15 [1982]). "Gram
positive
bacteria" are bacteria which retain the primary dye used in the Gram stain,
causing the
stained cells to appear dark blue to purple under the microscope. "Gram
negative bacteria"
do not retain the primary dye used in the Gram stain, but are stained by the
counterstain.
Thus, gram negative bacteria appear red.
As used herein, the term "microorganism" refers to any species or type of
microorganism, including but not limited to, bacteria, archaea, fungi,
protozoans,
mycoplasma, and parasitic organisms. The present invention contemplates that a
number of
microorganisms encompassed therein will also be pathogenic to a subject.
As used herein, the term "fungi" is used in reference to eukaryotic organisms
such as
the molds and yeasts, including dimorphic fungi.
As used herein, the term "virus" refers to minute infectious agents, which
with
certain exceptions, are not observable by light microscopy, lack independent
metabolism,
and are able to replicate only within a living host cell. The individual
particles (i. e., virions)
typically consist of nucleic acid and a protein shell or coat; some virions
also have a lipid
containing membrane. The term "virus" encompasses all types of viruses,
including animal,
plant, phage, and other viruses.
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The term "sample" as used herein is used in its broadest sense. A sample
suspected
of indicating a condition characterized by the dysregulation of apoptotic
function may
comprise a cell, tissue, or fluids, chromosomes isolated from a cell (e.g., a
spread of
metaphase chromosomes), genomic DNA (in solution or bound to a solid support
such as
for Southern blot analysis), RNA (in solution or bound to a solid support such
as for
Northern blot analysis), cDNA (in solution or bound to a solid support) and
the like. A
sample suspected of containing a protein may comprise a cell, a portion of a
tissue, an
extract containing one or more proteins and the like.
As used herein, the terms "purified" or "to purify" refer, to the removal of
undesired
components from a sample. As used herein, the term "substantially purified"
refers to
molecules that are at least 60% free, preferably 75% free, and most preferably
90%, or
more, free from other components with which they usually associated.
As used herein, the term "antigen binding protein" refers to proteins which
bind to a
specific antigen. "Antigen binding proteins" include, but are not limited to,
immunoglobulins, including polyclonal, monoclonal, chimeric, single chain, and
humanized
antibodies, Fab fragments, F(ab')2 fragments, and Fab expression libraries.
Various
procedures known in the art are used for the production of polyclonal
antibodies. For the
production of antibody, various host animals can be immunized by injection
with the
peptide corresponding to the desired epitope including but not limited to
rabbits, mice, rats,
sheep, goats, etc. In a preferred embodiment, the peptide is conjugated to an
immunogenic
carrier (e.g., diphtheria toxoid, bovine serum albumin (BSA), or keyhole
limpet hemocyanin
[KLH]). Various adjuvants are used to increase the immunological response,
depending on
the host species, including but not limited to Freund's (complete and
incomplete), mineral
gels such as aluminum hydroxide, surface active substances such as
lysolecithin, pluronic
polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,
dinitrophenol,
and potentially useful human adjuvants such as BCG (Bacille Calmette-Guerin)
and
Corynebacterium parvum.
For preparation of monoclonal antibodies, any technique that provides for the
production of antibody molecules by continuous cell lines in culture may be
used (See e.g.,
Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory Press,
Cold Spring Harbor, NY). These include, but are not limited to, the hybridoma
technique
originally developed by Kohler and Milstein (Kohler and Milstein, Nature,
256:495-497
CA 02524394 2008-11-13
[ 1975]), as well as the trioma technique, the human B-cell hybridoma
technique (See e.g.,
Kozbor et al., Immunol. Today, 4:72 [1983]), and the EBV-hybridoma technique
to produce
human monoclonal antibodies (Cole et al., in Monoclonal Antibodies and Cancer
Therapy,
Alan R. Liss, hic., pp. 77-96 [1985]).
According to the invention, techniques described for the production of single
chain
antibodies (U.S. 4,946,778 ) can be adapted to produce
specific single chain antibodies as desired. An additional embodiment of the
invention
utilizes the techniques known in the art for the construction of Fab
expression libraries
(Huse et al., Science, 246:1275-1281 [1989]) to allow rapid and easy
identification of
monoclonal Fab fragments with the desired specificity.
Antibody fragments that contain the idiotype (antigen binding region) of the
antibody molecule can be generated by known techniques. For example, such
fragments
include but are not limited to: the F(ab')2 fragment that can be produced by
pepsin digestion
of an antibody molecule; the Fab' fragments that can be generated by reducing
the disulfide
bridges of an F(ab')2 fragment, and the Fab fragments that can be generated by
treating an
antibody molecule with papain and a reducing agent.
Genes encoding antigen binding proteins can be isolated by methods known in
the
art. In the production of antibodies, screening for the desired antibody can
be accomplished
by techniques known in the art (e.g., radioimmunoassay, ELISA (enzyme-linked
immunosorbant assay), "sandwich" immunoassays, immunoradiometric assays, gel
diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays
(using
colloidal gold, enzyme or radioisotope labels, for example), Western Blots,
precipitation
reactions, agglutination assays (e.g., gel agglutination assays,
hemagglutination assays,
etc.), complement fixation assays, immunofluorescence assays, protein A
assays, and
immunoelectrophoresis assays, etc.) etc.
As used herein, the term "immunoglobulin" or "antibody" refer to proteins that
bind
a specific antigen. Immunoglobulins include, but are not limited to,
polyclonal,
monoclonal, chimeric, and humanized antibodies, Fab fragments, F(ab')2
fragments, and
includes in-ununoglobulins of the following classes: IgG, IgA, IgM, IgD, IbE,
and secreted
immunoglobulins (slg). Immunoglobulins generally comprise two identical heavy
chains
and two light chains. However, the terms "antibody" and "inununoglobulin" also
encompass single chain antibodies and two chain antibodies.
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The term "epitope" as used herein refers to that portion of an antigen that
makes
contact with a particular immunoglobulin. When a protein or fragment of a
protein is used
to immunize a host animal, numerous regions of the protein may induce the
production of
antibodies which bind specifically to a given region or three-dimensional
structure on the
protein; these regions or structures are referred to as "antigenic
determinants". An antigenic
determinant may compete with the intact antigen (i.e., the "immunogen" used to
elicit the
immune response) for binding to an antibody.
The terms "specific binding" or "specifically binding" when used in reference
to the
interaction of an antibody and a protein or peptide means that the interaction
is dependent
upon the presence of a particular structure (i.e., the antigenic determinant
or epitope) on the
protein; in other words the antibody is recognizing and binding to a specific
protein
structure rather than to proteins in general. For example, if an antibody is
specific for
epitope "A," the presence of a protein containing epitope A (or free,
unlabelled A) in a
reaction containing labeled "A" and the antibody will reduce the amount of
labeled A bound
to the antibody.
As used herein, the terms "non-specific binding" and "background binding" when
used in reference to the interaction of an antibody and a protein or peptide
refer to an
interaction that is not dependent on the presence of a particular structure
(i.e., the antibody
is binding to proteins in general rather that a particular structure such as
an epitope).
As used herein, the term "modulate" refers to the activity of a compound
(e.g.,
benzodiazepine compound) to affect (e.g., to promote or retard) an aspect of
cellular
function, including, but not limited to, cell growth, proliferation,
apoptosis, and the like.
As used herein, the term "competes for binding" is used in reference to a
first
molecule (e.g., a first benzodiazepine derivative) with an activity that binds
to the same
substrate (e.g., the oligomycin sensitivity conferring protein in
mitochondrial ATP synthase)
as does a second molecule (e.g., a second benzodiazepine derivative or other
molecule that
binds to the oligomycin sensitivity conferring protein in mitochondrial ATP
synthase, etc.).
The efficiency (e.g., kinetics or thermodynamics) of binding by the first
molecule may be
the same as, or greater than, or less than, the efficiency of the substrate
binding to the
second molecule. For example, the equilibrium binding constant (KD) for
binding to the
substrate may be different for the two molecules.
37
CA 02524394 2008-11-13
As used herein, the term "instructions for administering said
compound to a subject," and grammatical equivalents thereof, includes
instructions for
using the compositions contained in a kit for the treatment of conditions
characterized by
the dysregulation of apoptotic processes in a cell or tissue (e.g., providing
dosing, route of
administration, decision trees for treating physicians for correlating patient-
specific
characteristics with therapeutic courses of action). The term also
specifically refers to
instructions for using the compositions contained in the kit to treat
autoimmune disorders
(e.g., systemic lupus erythematosus, rheumatoid arthritis, graft-versus-host
disease,
myasthenia gravis, Sjogren's syndrome, etc.), chronic inflammatory conditions
(e.g.,
psoriasis, asthma and Crohn's disease), hyperproliferative disorders (e.g.,
tumors, B cell
lymphomas, T cell lymphoinas, etc.), viral infections (e.g., herpes virus,
papilloma virus,
HIV), and other conditions such as osteoarthritis and atherosclerosis, and the
like.
The term "test compound" refers to any chemical entity, pharmaceutical, drug,
and
the like, that can be used to treat or prevent a disease, illness, sickness,
or disorder of bodily
function, or otherwise alter the physiological or cellular status of a sample
(e.g., the level of
dysregulation of apoptosis in a cell or tissue). Test compounds comprise both
known and
potential therapeutic compounds. A test compound can be determined to be
therapeutic by
using the screening methods of the present invention. A "known therapeutic
compound"
refers to a therapeutic compound that has been shown (e.g., through animal
trials or prior
experience with administration to humans) to be effective in such treatment or
prevention.
In preferred embodiments, "test compounds" are agents that modulate apoptosis
in cells.
As used herein, the term "third party" refers to any entity engaged in
selling,
warehousing, distributing, or offering for sale a test compound contemplated
for
administered with a compound for treating conditions characterized by the
dysregulation of
apoptotic processes.
GENERAL DESCRIPTION OF THE INVENTION
As a class of drugs, benzodiazepine compounds have been widely studied and
reported to be effective medicaments for treating a number of disease. For
example, U.S.
4,076823, 4,110,337, 4,495,101, 4,751,223 and 5,776,946,
report that certain benzodiazepine compounds are effective as
analgesic and anti-inflainmatory agents. Similarly, U.S. 5,324,726 and U.S.
5,597,915,
38
CA 02524394 2008-11-13
report that certain benzodiazepine compounds
are antagonists of cholecystokinin and gastrin and thus might be useful to
treat certain
gastrointestinal disorders.
Other benzodiazepine compounds have been studied as inhibitors of human
neutrophil elastase in the treating of human neutrophil elastase-mediated
conditions such as
myocardial ischenua, septic shock syndrome, among others (See e.g., U.S.
5,861,380)
U.S. _5041,438,.
reports that certain benzodiazepine compounds are useful as anti-
retroviral agents.
Despite the attention benzodiazepine compounds have drawn, it will become
apparent from the description below, that the present invention provides novel
benzodiazepine compounds and related compounds and methods of using the novel
compounds, as well as known compounds, for treating a variety of diseases.
Benzodiazepine compounds are known to bind to benzodiazepine receptors in the
central nervous system (CNS) and thus have been used to treat various CNS
disorders
including anxiety and epilepsy. Peripheral benzodiazepine receptors have also
been
identified, which receptors may incidentally also be present in the CNS. The
present
invention demonstrates that benzodiazepines and related compounds have pro-
apoptotic and
cytotoxic properties useful in the treatment of transformed cells grown in
tissue culture.
The route of action of these compounds is not through the previously
identified
benzodiazepine receptors.
Experiments conducted during the development of the present invention have
identified novel biological targets for benzodiazepine compounds and related
compounds
(some of which are related by their ability to bind cellular target molecules
rather than their
homology to the overall chemical structure of benzodiazepine compounds). In
particular,
the present invention provides compounds that interact, directly or
indirectly, with particular
mitochondrial proteins to elicit the desired biological effects.
Thus, in some embodiments, the present invention provides a number of novel
compounds and previously known compounds directed against novel cellular
targets to
achieve desired biological results. In other embodiments, the present
invention provides
methods for using such compounds to regulate biological processes. The present
invention
also provides drug-screening methods to identify and optimize compounds. The
present
39
CA 02524394 2008-11-13
invention further provides diagnostic markers for identifying diseases and
conditions, for
monitoring treatment regimens, and/or for identifying optimal therapeutic
courses of action.
These and other research and therapeutic utilities are described below.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides novel chemical compounds, methods for their
discovery, and their therapeutic use. In particular, the present invention
provides
benzodiazepine derivatives and related compounds and methods of using
benzodiazepine
derivatives and related compounds as therapeutic agents to treat a number of
conditions
associated with the faulty regulation of the processes of programmed cell
death,
autoinu unity, inflammation, and hyperproliferation, and the like.
Exemplary compositions and methods of the present invention are described in
more
detail in the following sections: I. Modulators Of Cell Death; U. Modulators
Of Cell
Growth And Proliferation; III. Expression Analysis Of Treated Cells; IV.
Exemplary
Compounds; V. Pharmaceutical Compositions, Formulations, And Exemplary
Administration Routes And Dosing Considerations; VI. Drug Screens; VII.
Therapeutic
Applications; and VIII. ATPase Inhibitors And Methods For Identifying
Therapeutic
Inhibitors.
The practice of the present invention employs, unless otherwise indicated,
conventional techniques of organic chemistry, pharmacology, molecular biology
(including
recombinant techniques), cell biology, biochemistry, and immunology, which are
within the
skill of the art. Such techniques are explained fully in the literature, such
as, "Molecular
cloning: a laboratory manual" Second Edition (Sambrook et al., 1989);
"Oligonucleotide
synthesis" (M.J. Gait, ed., 1984); "Animal cell culture" (R.I. Freshney, ed.,
1987); the series
"Methods in enzymology" (Academic Press, Inc.); "Handbook of experimental
immunology" (D,M. Weir & C.C. Blackwell, eds); "Gene transfer vectors for
mammalian
cells" (J.M. Miller & M.P. Calos, eds., 1987); "Current protocols in molecular
biology"
(F.M. Ausubel et al., eds., 1987, and periodic updates); "PCR: the polymerase
chain
reaction" (Mullis et al., eds., 1994); and "Current protocols in immunology"
(J.E. Coligan et
al,, eds,, 1991),
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1. Modulators of Cell Death
In preferred embodiments, the present invention regulates apoptosis through
the
exposure of cells to compounds. The effect of compounds can be measured by
detecting
any number of cellular changes. Cell death may be assayed as described herein
and in the
art. In preferred embodiments, cell lines are maintained under appropriate
cell culturing
conditions (e.g., gas (C02), temperature and media) for an appropriate period
of time to
attain exponential proliferation without density dependent constraints. Cell
number and or
viability are measured using standard techniques, such as trypan blue
exclusion/hemo-
cytometry, or MTT dye conversion assay. Alternatively, the cell maybe analyzed
for the
expression of genes or gene products associated with aberrations in apoptosis
or necrosis.
In preferred embodiments, exposing the present invention to a cell induces
apoptosis. In some embodiments, the present invention causes an initial
increase in cellular
ROS levels (e.g., 02-). In further embodiments, exposure of the compounds of
the present
invention to a cell causes an increase in cellular 02 levels. In still further
embodiments, the
increase in cellular 02- levels resulting from the compounds of the present
invention is
detectable with a redox-sensitive agent that reacts specifically with 02
(e.g.,
dihyroethedium (DHE)).
In other embodiments, increased cellular 02" levels resulting from compounds
of the
present invention diminish after a period of time (e.g., 10 minutes). In other
embodiments,
increased cellular 02- levels resulting from the compounds of the present
invention diminish
after a period of time and increase again at a later time (e.g., 10 hours). In
further
embodiments, increased cellular 02 levels resulting from the compounds of the
present
invention diminish at 1 hour and increase again after 4 hours. In preferred
embodiments, an
early increase in cellular 02_ levels, followed by a diminishing in cellular
02 levels,
followed by another increase in cellular 02 levels resulting from the
compounds of the
present invention is due to different cellular processes (e.g., bimodal
cellular mechanisms).
In some embodiments, the present invention causes a collapse of a cell's
mitochondrial AT,,,. In preferred embodiments, a collapse of a cell's
mitochondrial
0`F,,, resulting from the present invention is detectable with a mitochondria-
selective
potentiometric probe (e.g., DiOC6). In further embodiments, a collapse of a
cell's
mitochondrial A%, resulting from the present invention occurs after an initial
increase in
cellular 02- levels.
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In some embodiments, the present invention enables caspace activation. In
other
embodiments, the present invention causes the release of cytochrome c from
mitochondria.
In further embodiments, the present invention alters cystolic cytochrome c
levels. In still
other embodiments, altered cystolic cytochrome c levels resulting from the
present
invention are detectable with immunoblotting cytosolic fractions. In preferred
embodiments, diminished cystolic cytochrome c levels resulting from the
present invention
are detectable after a period of time (e.g., 10 hours). In further preferred
embodiments,
diminished cystolic cytochrome c levels resulting from the present invention
are detectable
after 5 hours.
In other embodiments, the present invention causes the opening of the
mitochondrial
PT pore. In preferred embodiments, the cellular release of cytochrome c
resulting from the
present invention is consistent with a collapse of mitochondrial AIP,,,. In
still further
preferred embodiments, the present invention causes an increase in cellular 02
levels after a
mitochondrial A',,, collapse and a release of cytochrome c. In further
preferred
embodiments, a rise in cellular 02 levels is caused by a mitochondrial A'',r,
collapse and
release of cytochrome c resulting from the present invention.
In other embodiments, the present invention causes cellular caspase
activation. In
preferred embodiments, caspase activation resulting from the present invention
is
measurable with a pan-caspase sensitive fluorescent substrate (e.g., FAM-VAD-
fink). In
still further embodiments, caspase activation resulting from the present
invention tracks
with a collapse of mitochondrial i'I',,,. In other embodiments, the present
invention causes
an appearance of hypodiploid DNA. In preferred embodiments, an appearance of
hypodiploid DNA resulting from the present invention is slightly delayed with
respect to
caspase activation.
In some embodiments, the molecular target for the present invention is found
within
mitochondria. In further embodiments, the molecular target of the present
invention
involves the mitochondrial ATPase. The primary sources of cellular ROS include
redox
enzymes and the mitochondrial respiratory chain (hereinafter MRC). In
preferred
embodiments, cytochrome c oxidase (complex IV of the MRC) inhibitors (e.g.,
NaN3)
preclude a present invention dependent increase in cellular ROS levels. In
other preferred
embodiments, the ubiquinol-cytochrome c reductase component of MRC complex III
inhibitors (e.g., FK506) preclude a present invention dependent increase in
ROS levels.
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In some embodiments, an increase in cellular ROS levels due to the compounds
of
the present invention result from the binding of the compounds of the present
invention to a
target within mitochondria. In preferred embodiments, the compounds of the
present
invention oxidizes 2',7'-dichlorodihydrofluorescin (hereinafter DCF) diacetate
to DCF.
DCF is a redox-active species capable of generating ROS. In further
embodiments, the rate
of DCF production resulting from the present invention increases after a lag
period.
Antimycin A generates 02 by inhibiting ubiquinol-cytochrome c reductase. In
preferred embodiments, the present invention increases the rate of ROS
production in an
equivalent manner to antimycin A. In further embodiments, the present
invention increases
the rate of ROS production in an equivalent manner to antimycin A under
aerobic
conditions supporting state 3 respiration. In further embodiments, the
compounds of the
present invention do not directly target the MPT pore. In additional
embodiments, the
compounds of the present invention do not generate substantial ROS in the
subcellular S 15
fraction (e.g., cytosol; microsomes). In even further embodiments, the
compounds of the
present invention do not stimulate ROS if mitochondria are in state 4
respiration.
MRC complexes I - III are the primary sources of ROS within mitochondria. In
preferred embodiments, the primary source of an increase in cellular ROS
levels resulting
from the dependent invention emanates from these complexes as a result of
inhibiting the
mitochondrial F1F0-ATPase. Indeed, in still further embodiments, the present
invention
inhibits mitochondrial ATPase activity of bovine sub-mitochondrial particles
(hereinafter
SMPs). In particularly preferred embodiments, the compounds of the present
invention
bind to the OSCP component of the mitochondrial F1F0-ATPase.
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In some embodiments, the compounds of the present invention have the
structure:
R1
0
N
R4 R2
/ /N
\\R3
or
'
N
R4 R2
N
R3
or its enantiomer, wherein, R1 is aliphatic or aryl; R2 is aliphatic, aryl, -
NH2, -HC(=O)-R5,
or a moiety that participates in hydrogen bond formation, wherein R5 is aryl,
heterocyclic, -
R6-NH-C(=O)-R7 or -R6-C(=O)-NH-R7, wherein R6 is an aliphatic linker of 1-6
carbons and
R7 is aliphatic, aryl, or heterocyclic; and each of R3 and R4 is independently
hydrogen,
hydroxy, alkoxy, halo, amino, lower-alkyl-substituted-amino, acylamino,
hydroxyamino, an
aliphatic group having 1-8 carbons and 1-20 hydrogens, aryl, or heteroaryl; or
a
pharmaceutically acceptable salt, prodrug or derivative thereof. In some
preferred
embodiments, where R3 is a hydroxyl group, one or more additional positions on
the ring
containing R3 includes a chemical group (e.g., an alkyl chain) that protects
the hydroxyl
group from metabolism in vivo.
In certain embodiments, the compounds of the present invention may have a
hydroxyl group at the C'4 position and an aromatic ring. In preferred
embodiments,
compounds of the present invention cause an increase in cellular ROS levels as
a result of a
hydroxyl group at the C'4 position and an aromatic ring. In further
embodiments, the
potency of the present invention in cell based assays correlates with ATPase
inhibition
experiments using SMPs. Indeed, in preferred embodiments, the present
invention
significantly inhibits mitochondrial ATPase activity in comparison to
cytotoxic (80 M)
concentrations of general benzodiazepines and PBR ligands (e.g., PK1 1195 and
4-
chlorodiazepam) that do not significantly inhibit mitochondrial ATPase
activity. As such,
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in preferred embodiments, the molecular target of the present invention is the
mitochondrial
ATPase.
Oligomycin is a macrolide natural product that binds to the mitochondrial F1F0-
ATPase, induces a state 3 to 4 transition, and as a result, generates ROS
(e.g., 02-)- In
preferred embodiments, the present invention binds the OSCP component of the
mitochondrial F1F0-ATPase. In certain embodiments, screening assays of the
present
invention permit detection of binding partners of the OSCP. OSCP is an
intrinsically
fluorescent protein. In certain embodiments, titrating a solution of test
compounds of the
present invention into an E. Coli sample overexpressed with OSCP results in
quenching of
the intrinsic OSCP fluorescence. In other embodiments, fluorescent or
radioactive test
compounds can be used in direct binding assays. In other embodiments,
competition
binding experiments can be conducted. In this type of assay, test compounds
are assessed
for their ability to compete with Bz-423 for binding to the OSCP. In some
embodiments,
the compounds of the present invention cause a reduced increase in cellular
ROS levels and
reduced apoptosis in cells through regulation of the OSCP gene (e.g., altering
expression of
the OSCP gene). In further embodiments, the present invention functions by
altering the
molecular motions of the ATPase motor.
II. Modulators of Cellular Proliferation and Cell Growth
In some embodiments, the compounds and methods of the present invention cause
descreased cellular proliferation. In other embodiments, the compounds and
methods of the
present invention causes decreased cellular proliferation and apoptosis. For
example, cell
culture cytotoxicity assays conducted during the development of the present
invention
demonstrated that the compounds and methods of the present invention prevents
cell growth
after an extended period in culture (e.g., 3 days).
III. Expression Analysis of Treated Cells
During the development of the present invention, an expression profile was
generated to identify those genes that are differentially expressed in treated
and untreated
cells. This profile provides a gene expression fingerprint of cells induced by
the compounds
of the present invention. This fingerprint identifies genes that are
upregulated and
downregulated in response to the compounds of the present invention and
identifies such
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genes are diagnostic markers for drug screening and for monitoring therapeutic
effects of
the compounds. The genes also provide targets for regulation to mimic the
effects of the
compounds of the present invention. Data from an expression analysis for genes
up-
regulated in the presence of Bz-423 is presented in Figure 4A. Data from an
expression
analysis for genes down-regulated in the presence of Bz-423 is presented in
Figure 4B.
Data from an expression analysis for genes up-regulated in the presence of Bz-
OMe is
presented in Figure 4C. Data from an expression analysis for genes down-
regulated in the
presence of Bz-OMe is presented in Figure 4D.
For example, an analysis of the expression profile provides ornithine
decarboxylase
antizyme 1 (OAZ1) as a novel therapeutic agent. OAZ1 is an important
regulatory protein
that controls the synthesis and transport into cells of polyamines, including
putrescine,
spermidine and spermine. The synthesis of poylamines in cells involves several
enzymatic
steps, however ornithine decarboxylase is the enzyme that principally
regulates this process.
By inhibiting the polyamine transporter located in the plasma membrane and by
targeting
ornithine decarboxylase for proteolytic degradation, OAZ1 reduces polyamine
levels in
cells. Polyamines are essential for the survival and growth of cells. Abnormal
accumulation of polyamines contributes to tumor induction, cancer growth and
metastasis.
Inhibitors of polyamine biosynthesis, and specifically one molecule identified
as
difluoromethylornithine (DFMO), are in clinical trials to confirm their
anticarcinogenic and
therapeutic potential. In preferred embodiments of the present invention, OAZI
is induced
to a level 16-fold above the level of control cells in cells treated with the
compounds of the
present invention. Any method, direct or indirect, for inducing OAZ1 levels is
contemplated by the present invention (e.g., treatment with compounds of the
present
invention, gene therapy, etc.).
OAZ1 is an important regulator of polyamine metabolism and functions to
decrease
polyamine levels by acting as an inhibitor of ornithine decarboxylase (ODC), a
mitochondrial enzyme that controls the rate-limiting step of polyamine
biosynthesis. After
inhibition with antizyme, ODC is targeted for proteosomal degredation.
Polyamines are
intimately involved in cellular stability and required for cell proliferation.
Inhibiting
polyamine synthesis suppresses proliferation. As such, in still further
embodiments, ODC
expression or activity is decreased (e.g., using siRNA, antisense
oligonucleotides, gene
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therapy, known or later identified inhibitors, the compounds of the present
invention, etc.)
to elicit the desired biological effect.
Antizyme 1 expression is regulated transcriptionally and at the post-
transcriptional
level. Post-transcriptional regulation plays a particularly important role in
the regulation of
this gene product and occurs by a unique translational frameshift that depends
on either
polymanes (through a negative-feedback loop) or agmatine, another metabolite
of arginine.
ODC activity leves may be obtained by quanifying the conversion of ornithine
to putrescine
using 3H-ornithine. In some embodiments, treating cells with the compounds of
the present
invention significantly reduces ODC activity in a dose-dependant fashion. In
still further
embodiments, a reduction in ODC activity is paralleled by a decrease in ODC
protein levels
measured under similar conditions. Cells pre-incubated with MnTBAP decrease
ROS
levels. In some embodiments, cells pre-incubated with MnTBAP that are exposed
to the
compounds of the present invention display reversed inhibition of ODC.
In preferred embodiments, cells treated with high levels (e.g., >10 M) of the
compounds of the present invention generate sufficient amounts of ROS that are
not
detoxified by cellular anti-oxidants, and result in apoptosis within a short
time period (e.g.,
18 h). In preferred embodiments, cells treated with lower levels (e.g., <10
M) of the
compounds of the present invention induce a reduced ROS response that is
insufficient to
trigger apoptosis, but is capable of inhibiting ODC or otherwise blocking
cellular
proliferation. In other embodiments, a derivative of the compounds of the
present invention
in which the phenolic hydroxyl is replaced by Cl or OCH3 is minimally
cytotoxic, generates
a small ROS response in cells, binds less tightly to the OSCP, and inhibits
ODC activity. In
still other embodiments, cells treated with a derivative of the compounds of
the present
invention in which the phenolic hydroxyl is replaced by Cl experience reduced
proliferation
to a similar extent as to the unmodified compounds. As such, in preferred
embodiments, the
antiproliferative effects are obtained using chemical derivatives of the
compounds of the
present invention that block proliferation without inducing apoptosis.
In response to antigenic or mitogenic stimulation, lymphocytes secrete protein
mediators, one of which is named migration inhibitory factor (MIF) for its
ability to prevent
the migration of macrophages in vitro. MIF maybe an anti-tumor agent. In
addition, the
ability of MIF to prevent the migration of macrophages may be exploited for
treating
wounds. MIF may alter the immune response to different antigens. MIF links
chemical and
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immunological detoxification systems. MIF was induced approximately 10-fold by
Bz-423.
Thus, the present invention contemplates the use of MIF as a target of the
compounds of the
present invention.
Prolifin is induced at high levels in cell treated with the present invention.
Profilin
binds to actin monomers and interacts with several proteins and
phosphoinositides, linking
signaling pathways to the cytoskeleton. Profilin can sequester actin monomers,
increase
exchange of ATP for ADP on actin, and increase the rate of actin filament
turnover. A
comparison between several different tumorigenic cancer cell lines with
nontumorigenic
lines show consistently lower profilin 1 levels in tumor cells. Transfection
of profilin 1
cDNA into CAL51 breast cancer cells raised the profilin 1 level, had a
prominent effect on
cell growth, and suppressed tumorigenicity of the overexpressing cell clones
in nude mice.
Therefore, induction of profilin 1 (e.g., by the compounds of the present
invention or
otherwise) may suppress the tumorigenesis of cancer cells.
Interferon regulatory factor 4 (IRF-4) is induced at higher than normal levels
in cells
treated with the compounds of the present invention. IRF-4 is a
lymphoid/myeloid-
restricted member of the IRF transcription factor family that plays an
essential role in the
homeostasis and function of mature lymphocytes. IRF-4 expression is regulated
in resting
primary T cells and is transiently induced at the mRNA and protein levels
after activation
by stimuli such as TCR cross-linking or treatment with phorbol ester and
calcium ionophore
(PMA/ionomycin). Stable expression of IRF-4 in Jurkat cells leads to a strong
enhancement
in the synthesis of interleukin (IL)-2, IL-4, IL- 10, and IL- 13. IRF-4
represents one of the
lymphoid-specific components that control the ability of T lymphocytes to
produce a
distinctive array of cytokines. In Abelson-transformed pro-B cell lines,
enforced expression
of IRF-4 is sufficient to induce germline Igk transcription. The action of the
compounds of
the present invention to induce IRF-4 may account for its affects on
autoimmune disease in
B and T cell dominant processes as well as for its ability to influence the
survival of
neoplastic B cell clones. In preferred embodiments, cell death-regulatory
protein GRIM 19
is induced at higher than normal levels in cells treated with the compounds of
the present
invention. The importance of the interferon (IFN) pathway in cell growth
suppression is
known. Studies have shown that a combination of IFN and all-trans retinoic
acid inhibits
cell growth in vitro and in vivo more potently than either agent alone. The
specific genes
that play a role in IFN/RA-induced cell death were identified by an antisense
knockout
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approach, and called GRIM genes. GRIM19 is a novel cell death-associated gene
that is not
included in any of the known death gene categories. This gene encodes a 144-aa
protein that
localizes to the nucleus. Overexpression of GRIM 19 enhances caspase-9
activity and
apoptotic cell death in response to IFN/RA treatment. GRIM19 is located in the
19p 13.2
region of the human chromosome essential for prostate tumor suppression,
signifying that
the protein may be a novel tumor suppressor. The induction of GRIM19 by the
compounds
of the present invention may result in anti-tumor effects.
IV. Exemplary Compounds
Exemplary compounds of the present invention are provided below.
R1
O
R4 R2
N
R3
or
R1
0
N
R4 R2
N
R3
O
or its enantiomer, wherein, R1 is aliphatic or aryl; R2 is aliphatic, aryl, -
NH2, -NHC(=O)-R5;
or a moiety that participates in hydrogen bonding, wherein R5 is aryl,
heterocyclic, -R6-NH-
C(=O)-R7 or -R6-C(=O)-NH-R7, wherein R6 is an aliphatic linker of 1-6 carbons
and R7 is
aliphatic, aryl, or heterocyclic, each of R3 and R4 is independently a
hydroxy, alkoxy, halo,
amino, lower-alkyl-substituted-amino, acetylamino, hydroxyamino, an aliphatic
group
having 1-8 carbons and 1-20 hydrogens, aryl, or heterocyclic; or a
pharmaceutically
acceptable salt, prodrug or derivative thereof.
In the above structures, Rl is a hydrocarbyl group of 1-20 carbons and 1-20
hydrogens. Preferably, Rl has 1-15 carbons, and more preferably, has 1-12
carbons.
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Preferably, Rl has 1-12 hydrogens, and more preferably, 1-10 hydrogens. Thus
Rl can be
an aliphatic group or an aryl group.
The term "aliphatic" represents the groups commonly known as alkyl, alkenyl,
alkynyl, alicyclic. The term "aryl" as used herein represents a single
aromatic ring such as a
phenyl ring, or two or more aromatic rings that are connected to each other
(e.g., bisphenyl)
or fused together (e.g., naphthalene or anthracene). The aryl group can be
optionally
substituted with a lower aliphatic group (e.g., C1-C4 alkyl, alkenyl, alkynyl,
or C3-C6
alicyclic). Additionally, the aliphatic and aryl groups can be further
substituted by one or
more functional groups such as -NH2, -NHCOCH3, -OH, lower alkoxy (Cl-C4), halo
(-F, -
Cl, -Br, or -I). It is preferable that Rl is primarily a nonpolar moiety.
In the above structures, R2 can be aliphatic, aryl, -NH2, -NHC(=O)-R5, or a
moiety
that participates in hydrogen bonding, wherein R5, is aryl, heterocyclic, R6-
NH-C(=O)-R7 or
-R6-C(=O)-NH-R7, wherein R6 is an aliphatic linker of 1-6 carbons and R7 is an
aliphatic,
aryl, or heterocyclic. The terms "aliphatic" and "aryl" are as defined above.
The term "a moiety that participates in hydrogen bonding" as used herein
represents
a group that can accept or donate a proton to form a hydrogen bond thereby.
Some specific non-limiting examples of moieties that participate in hydrogen
bonding include a fluoro, oxygen-containing and nitrogen-containing groups
that are well-
known in the art. Some examples of oxygen-containing groups that participate
in hydrogen
bonding include: hydroxy, lower alkoxy, lower carbonyl, lower carboxyl, lower
ethers and
phenolic groups. The qualifier "lower" as used herein refers to lower
aliphatic groups (Cl-
C4) to which the respective oxygen-containing functional group is attached.
Thus, for example, the term "lower carbonyl" refers to inter alia,
formaldehyde,
acetaldehyde.
Some nonlimiting examples of nitrogen-containing groups that participate in
hydrogen bond formation include amino and amido groups. Additionally, groups
containing both an oxygen and a nitrogen atom can also participate in hydrogen
bond
formation. Examples of such groups include nitro, N-hydroxy and nitrous
groups.
It is also possible that the hydrogen-bond acceptor in the present invention
can be
the fl electrons of an aromatic ring. However, the hydrogen bond participants
of this
invention do not include those groups containing metal atoms such as boron.
Further the
hydrogen bonds formed within the scope of practicing this invention do not
include those
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formed between two hydrogens, known as "dihydrogen bonds." (See, R.H.
Crabtree,
Science, 282:2000-2001 [1998], for further description of such dihydrogen
bonds).
The term "heterocyclic" represents, for example, a 3-6 membered aromatic or
nonaromatic ring containing one or more heteroatoms. The heteroatoms can be
the same or
different from each other. Preferably, at least one of the heteroatom's is
nitrogen. Other
heteroatoms that can be present on the heterocyclic ring include oxygen and
sulfur.
Aromatic and nonaromatic heterocyclic rings are well-known in the art. Some
nonlimiting examples of aromatic heterocyclic rings include pyridine,
pyrimidine, indole,
purine, quinoline and isoquinoline. Nonlimiting examples of nonaromatic
heterocyclic
compounds inciude piperidine, piperazine, morpholine, pyrrolidine and
pyrazolidine.
Examples of oxygen containing heterocyclic rings include, but not limited to
furan, oxirane,
2H-pyran, 4H-pyran, 2H-chromene, and benzofuran. Examples of sulfur-containing
heterocyclic rings include, but are not limited to, thiophene, benzothiophene,
and
parathiazine.
Examples of nitrogen containing rings include, but not limited to, pyrrole,
pyrrolidine, pyrazole, pyrazolidine, imidazole, imidazoline, imidazolidine,
pyridine,
piperidine, pyrazine, piperazine, pyrimidine, indole, purine, benzimidazole,
quinoline,
isoquinoline, triazole, and triazine.
Examples of heterocyclic rings containing two different heteroatoms include,
but are
not limited to, phenothiazine, morpholine, parathiazine, oxazine, oxazole,
thiazine, and
thiazole.
The heterocyclic ring is optionally further substituted with one or more
groups
selected from aliphatic, nitro, acetyl (i.e., -C(=O)-CH3), or aryl groups.
Each of R3 and R4 can be independently a hydroxy, alkoxy, halo, amino, or
substituted amino (such as lower-alkyl-substituted-amino, or acetylamino or
hydroxyamino), or an aliphatic group having 1-8 carbons and 1-20 hydrogens.
When each
of R3 and R4 is an aliphatic group, it can be further substituted with one or
more functional
groups such as a hydroxy, alkoxy, halo, amino or substituted amino groups as
described
above. The terms "aliphatic" is defined above. Alternatively, each of R3 and
R4 can be
hydrogen.
It is well-known that many 1,4-benzodiazepines exist as optical isomers due to
the
chirality introduced into the heterocyclic ring at tile C3 position. The
optical isomers are
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sometimes described as L- or D-isomers in the literature. Alternatively, the
isomers are also
referred to as R- and S- enantiomorphs. For the sake of simplicity, these
isomers are
referred to as enantiomorphs or enantiomers. The 1,4-benzodiazepine compounds
described
herein include their enantiomeric forms as well as racemic mixtures. Thus, the
usage
"benzodiazepine or its enantiomers" herein refers to the benzodiazepine as
described or
depicted, including all its enantiomorphs as well as their racemic mixture.
From the above description, it is apparent that many specific examples are
represented by the generic formulas presented above. Thus, in one example, Rl
is aliphatic,
R2 is aliphatic, whereas in another example, Rl is aryl and R2 is a moiety
that participates in
hydrogen bond formation. Alternatively, Rl can be aliphatic, and R2 can be an -
NHC(=O)-
R5, or a moiety that participates in hydrogen bonding, wherein R5 is aryl,
heterocyclic, -R6-
NH-C(=O)-R7 or -R6-C(=O)-NH-R7, wherein R6 is an aliphatic linker of 1-6
carbons and R7
is an aliphatic, aryl, or heterocyclic. A wide variety of sub combinations
arising from
selecting a particular group at each substituent position are possible and all
such
combinations are within the scope of this invention.
Further, it should be understood that the numerical ranges given throughout
this
disclosure should be construed as a flexible range that contemplates any
possible subrange
within that range. For example, the description of a group having the range of
1-10 carbons
would also contemplate a group possessing a subrange of, for example, 1-3, 1-
5, 1-8, or 2-3,
2-5, 2-8, 3-4, 3-5, 3-7, 3-9, 3-10, etc., carbons. Thus, the range 1-10 should
be understood
to represent the outer boundaries of the range within which many possible
subranges are
clearly contemplated. Additional examples contemplating ranges in other
contexts can be
found throughout this disclosure wherein such ranges include analogous
subranges within.
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Some specific examples of the benzodiazepine compounds of this invention
include:
H3C
O H O
N N 7
R2 R2
CI N CI N
HO HO
H3C O
O
N
H
N
R2
R2
CI N
CI N
CH3 O\
CH3
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wherein R2 is
I
OH
I I
N
N
and
N
and dimethylphenyl (all isomers) and ditrifluoromethyl (all isomers).
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The following compounds are also contemplated:
NHa
CI
HO
H-Biotin
C11 HO
This invention also provides the compound Bz-423.
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CH3
CI N
CH3
OH
Bz-423 differs from benzodiazepines in clinical use by the presence of a
hydrophobic substituent at C-3. This substitution renders binding to the
peripheral
benzodiazepine receptor ("PBR") weak (Id ca. 1 M) and prevents binding to the
central
benzodiazepine receptor so that Bz-423 is not a sedative.
In some embodiments R2 is any chemical group that permits the compound to bind
to OSCP. In some such embodiments, R2 comprises a hydrophobic aromatic group.
In
preferred embodiments R2 comprises a hydrophobic aromatic group larger than
benzene
(e.g., a benzene ring with non-hydrogen substituents, a moiety having two or
more aromatic
rings, a moiety with 7 or more carbon atoms, etc.).
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Additional specific benzodiazepine derivative examples of the present
invention
include the following:
0
R5 R1 = H, alkyl, or substituted alkyl
6R, R3 = H, alkyl, or substituted alkyl
R4 = H, alkyl, or substituted alkyl
/ sterochemistry is R, S, or racemic
R2 is selected from hydrogen, a hydroxy, an alkoxy, a
halo, an amino, a lower-alkyl-a substituted-amino, an
acetylamino, a hydroxyamino, an aliphatic group having
R 1-8 carbons and 1-20 hydrogens, a substituted aliphatic
4 group of similar size, a cycloaliphatic group consisting of
R2 < 10 carbons, a substituted cycloaliphatic group, an aryl,
R3 OH and a heterocyclic
R5 = + -~ -~
(CH2)nC(CH3)3 (CH2)nCH(CH3)2 CH2(CH2)nCH3
n=0-5 n0-5 n=0-5
dialkyl (all regioisomers)
i
01/
difluoromethyl (all regioisomers)
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0
R5 R1 = H, alkyl, or substituted alkyl
6R, R3 = H, alkyl, or substituted alkyl
R4 = H, alkyl, or substituted alkyl
/ sterochemistry is R, S, or racemic
R2 is selected from hydrogen, a hydroxy, an alkoxy, a
halo, an amino, a lower-alkyl-a substituted-amino, an
acetylamino, a hydroxyamino, an aliphatic group having
R 1-8 carbons and 1-20 hydrogens, a substituted aliphatic
4 group of similar size, a cycloaliphatic group consisting of
R2 < 10 carbons, a substituted cycloaliphatic group, an aryl,
R3 OH and a heterocyclic
R5 = + +
(CH2)nC(CH3)3 (CH2)nCH(CH3)2 CH2(CH2)nCH3
n=0-5 n=0-5 n=0-5
dialkyl (all regioisomers)
vwL
difluoromethyl (all regioisomers)
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R4
R2 = H, alkyl, or substituted alkyl
R3 = H, alkyl, or substituted alkyl
Rl is selected hydrogen, a hydroxy, an alkoxy, a halo, an
amino, a lower-alkyl-a substituted-amino, an acetylamino,
a hydroxyamino, an aliphatic group having 1-8 carbons
R and 1-20 hydrogens, a substituted aliphatic group of
3 similar size, a cycloaliphatic group consisting of < 10
R1 carbons, a substituted cycloaliphatic group, an aryl, and a
R2 OH heterocyclic
R4 = - - -
(CH2)nC(CH3)3 (CH2)nCH(CH3)2 CH2(CH2)nCH3
n=0-5 n=0-5 n=0-5
dialkyl (all regioisomers)
difluoromethyl (all regioisomers)
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R3
R1 = H, alkyl, or substituted alkyl
R2 = H, alkyl, or substituted alkyl
/ \
R2
R, OH
R3 = - - _
(CH2)õ C(CH3)3 (CH2)õ CH(CH3)2 CH2(CH2)õ CH3
n=0-5 n0-5 n=0-5
dialkyl (all regioisomers)
i
01/
difluoromethyl (all regioisomers)
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kR3
R1 = H, alkyl, or substituted alkyl
R2 = H, alkyl, or substituted alkyl
/ \
R2
R, OH
R3 = - - -
(CH2)nC(CH3)3 (CH2)nCH(CH3)2 CH2(CH2)nCH3
n=0-5 n=0-5 n=0-5
dialkyl (all regioisomers)
-
difluoromethyl (all regioisomers)
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0
R3 R1 = H, alkyl, or substituted alkyl
RjN R2 = H, alkyl, or substituted alkyl
R2 sterochemistry is R, S, or racemic
0
R3 = - - -
I I I
(CH2)nC(CH3)3 (CH2)nCH(CH3)2 CH2(CH2)nCH3
n=0-5 n0-5 n=0-5
dialkyl (all regioisomers)
\I/
difluoromethyl (all regioisomers)
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0
R5 R1 = H, alkyl, or substituted alkyl
RjN R3 = H, alkyl, or substituted alkyl
R4 = H, alkyl, or substituted alkyl
N
sterochemistry is R, S, or racemic
R4
R2 is selected from hydrogen, hydroxy, an alkoxy, a halo,
an amino, a lower-alkyl-a substituted-amino, an
acetylamino, a hydroxyamino, an aliphatic group having
OH 1-8 carbons and 1-20 hydrogens, a substituted aliphatic
R group of similar size, a cycloaliphatic group consisting of
2 < 10 carbons, a substituted cycloaliphatic group, an aryl,
R3 and a heterocyclic
R5 = - - -
(CH2)r,C(CH3)3 (CH2)õ CH(CH3)2 CH2(CH2)r,CH3
n=0-5 n=0-5 n=0-5
dialkyl (all regioisomers)
difluoromethyl (all regioisomers)
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0
R5 RI = H, alkyl, or substituted alkyl
R1 N R3 = H, alkyl, or substituted alkyl
R4 = H, alkyl, or substituted alkyl
/ sterochemistry is R, S, or racemic
/ R4
\ R2 is selected from hydrogen, hydroxy, an alkoxy, a halo,
/ \ an amino, a lower-alkyl-a substituted-amino, an
acetylamino, a hydroxyamino, an aliphatic group having
OH 1-8 carbons and 1-20 hydrogens, a substituted aliphatic
R group of similar size, a cycloaliphatic group consisting of
2 < 10 carbons, a substituted cycloaliphatic group, an aryl,
R3 and a heterocyclic
R5 A
(CH2)nC(CH3)3 (CH2)nCH(CH3)2 CH2(CH2)nCH3
n=0-5 n=0-5 n=0-5
-- \ / \
dialkyl (all regioisomers)
difluoromethyl (all regioisomers)
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0
R4 R1 = H, alkyl, or substituted alkyl
R1N R3 = H, alkyl, or substituted alkyl
N
sterochemistry is R, S, or racemic
/ \ R2 is selected from hydrogen, hydroxy, an allcoxy, a halo,
an amino, a lower-alkyl-a substituted-amino, an
acetylamino, a hydroxyamino, an aliphatic group having
R 1-8 carbons and 1-20 hydrogens, a substituted aliphatic
3 group of similar size, a cycloaliphatic group consisting of
R2 < 10 carbons, a substituted cycloaliphatic group, an aryl,
and a heterocyclic
R4 = -I - -
(CH2)nC(CH3)3 (CH2)nCH(CH3)2 CH2(CH2)nCH3
n=0-5 n=0-5 n=0-5
-- \ / \
dialkyl (all regioisomers)
difluoromethyl (all regioisomers)
\ / \
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0
R4 R1 = H, alkyl, or substituted alkyl
6R, R3 = H, alkyl, or substituted alkyl
/ sterochemistry OH is R, S, or racemic
H
R2 is selected from hydrogen, hydroxy, an alkoxy, a halo,
an amino, a lower-alkyl-a substituted-amino, an
acetylamino, a hydroxyamino, an aliphatic group having
R 1-8 carbons and 1-20 hydrogens, a substituted aliphatic
3 group of similar size, a cycloaliphatic group consisting of
R2 < 10 carbons, a substituted cycloaliphatic group, an aryl,
and a heterocyclic
R4 = - - -
(CH2)nC(CH3)3 (CH2)nCH(CH3)2 CH2(CH2)nCH3
n=0-5 n=0-5 n=0-5
dialkyl (all regioisomers)
i
01/
difluoromethyl (all regioisomers)
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OH
R3 R6 R5
HOR 2 / OH
I
R4
R1
R1 is H or hydroxy
Each of R2 through R6 may be the same or different and is selected from
hydrogen, a hydroxy,
an alkoxy, a halo, an amino, a lower-alkyl-a substituted-amino, an
acetylamino, a hydroxyamino,
an aliphatic group having 1-8 carbons and 1-20 hydrogens, a substituted
aliphatic group of
similar size, a cycloaliphatic group consisting of < 10 carbons, a substituted
cycloaliphatic
group, an aryl, and a heterocyclic
R8
R1 O Ry R7
R
2
R6
R
O
R3 R10
4
Each of R1 through R10 maybe the same or different and is selected from
hydrogen, a hydroxy,
an alkoxy, a halo, an amino, a lower-alkyl-a substituted-amino, an
acetylamino, a hydroxyamino,
an aliphatic group having 1-8 carbons and 1-20 hydrogens, a substituted
aliphatic group of
similar size, a cycloaliphatic group consisting of < 10 carbons, a substituted
cycloaliphatic
group, an aryl, and a heterocyclic
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O O
As
O
C02
NH
O 5
H
N
HN~~ H 0
\
O
As OH
HO
C02-
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R8
R9 R7
R2 O
R6
R5
R3 RIO
R4 R11
Each of Rl through Rl 1 maybe the same or different and is selected from
hydrogen, a hydroxy,
an alkoxy, a halo, an amino, a lower-alkyl-a substituted-amino, an
acetylamino, a hydroxyamino,
an aliphatic group having 1-8 carbons and 1-20 hydrogens, a substituted
aliphatic group of
similar size, a cycloaliphatic group consisting of < 10 carbons, a substituted
cycloaliphatic
group, an aryl, and a heterocyclic
R1 O
R2 RIO R
R$
3
R O
R4
R5 R7
R6
Each of Rl through R10 maybe the same or different and is selected from
hydrogen, a hydroxy,
an alkoxy, a halo, an amino, a lower-alkyl-a substituted-amino, an
acetylamino, a hydroxyamino,
an aliphatic group having 1-8 carbons and 1-20 hydrogens, a substituted
aliphatic group of
similar size, a cycloaliphatic group consisting of < 10 carbons, a substituted
cycloaliphatic
group, an aryl, and a heterocyclic
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OH
R1 H2C
R2
I \
N
R3 R5
R4
Each of R1 through R10 maybe the same or different and is selected from
hydrogen, a hydroxy,
an alkoxy, a halo, an amino, a lower-alkyl-a substituted-amino, an
acetylamino, a hydroxyamino,
an aliphatic group having 1-8 carbons and 1-20 hydrogens, a substituted
aliphatic group of
similar size, a cycloaliphatic group consisting of < 10 carbons, a substituted
cycloaliphatic
group, an aryl, and a heterocyclic
R, O O R6
H3CO OCH3
I
HO R3 R4 OH
R2 R5
Each of R1 through R6 maybe the same or different and is selected from
hydrogen, a hydroxy,
an alkoxy, a halo, an amino, a lower-alkyl-a substituted-amino, an
acetylamino, a hydroxyamino,
an aliphatic group having 1-8 carbons and 1-20 hydrogens, a substituted
aliphatic group of
similar size, a cycloaliphatic group consisting of < 10 carbons, a substituted
cycloaliphatic
group, an aryl, and a heterocyclic
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N
N O
N OCH2CF3
Iansoprazole
OH 0
O
HO
radicicol
CI
O
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0
R3 R1 = H, alkyl, or substituted alkyl
R1N R4 = H, alkyl, or substituted alkyl
R4 sterochemistry is R, S, or racemic
/ R2 is selected from hydrogen, a hydroxy, an alkoxy, a
\ 0 halo, an amino, a lower-alkyl-a substituted-amino, an
acetylamino, a hydroxyamino, an aliphatic group having
1-8 carbons and 1-20 hydrogens, a substituted aliphatic
group of similar size, a cycloaliphatic group consisting of
R2 < 10 carbons, a substituted cycloaliphatic group, an aryl,
and a heterocyclic
R3 = -1 A
(CH2)r,C(CH3)3 (CH2)nCH(CH3)2 CH2(CH2)r,CH3
n=0-5 n=0-5 n=0-5
A
dialkyl (all regioisomers)
4<\
difluoromethyl (all regioisomers)
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CH 3
O
R
1
CI N
OH
wherein R1 is selected from napthalalanine; phenol; 1-Napthalenol; 2-
Napthalenol;
Halogen
Halogen .
I I
OCF3 and
quinolines.
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A composition comprising the following formula:
CH3
O
CI N
R,
n ~rinti
wherein Rl is selected from: OH OH; CI
N3 OH ; and OCH3
The stereochemistry of all derivatives embodied in the present invention is R,
S, or racemic.
Additional specific benzodiazepine derivative examples of the present
invention
include the following:
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A composition, comprising the following formula:
H O
N
N
CI
HO
A composition comprising the following formula:
R7
\ O
N
R6 Ra
N
R,
R4
R2
R3
R5
wherein Rl, R2, R3 and R4 are selected from the group consisting of: hydrogen;
CH3; a
linear or branched, saturated or unsaturated aliphatic chain having at least 2
carbons; a
linear or branched, saturated or unsaturated aliphatic chain having at least 2
carbons, and
having at least one hydroxy subgroup; a linear or branched, saturated or
unsaturated
aliphatic chain having at least 2 carbons, and having at least one thiol
subgroup; a linear or
branched, saturated or unsaturated aliphatic chain having at least 2 carbons,
wherein said
aliphatic chain terminates with an aldehyde subgroup; a linear or branched,
saturated or
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unsaturated aliphatic chain having at least 2 carbons, and having at least one
ketone
subgroup; a linear or branched, saturated or unsaturated aliphatic chain
having at least 2
carbons; wherein said aliphatic chain terminates with a carboxylic acid
subgroup; a linear or
branched, saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at
least one amide subgroup; a linear or branched, saturated or unsaturated
aliphatic chain
having at least 2 carbons, and having at least one acyl group; a linear or
branched, saturated
or unsaturated aliphatic chain having at least 2 carbons, and having at least
one nitrogen
containing moiety (e.g.,nitro, nitrile, etc.); a linear or branched, saturated
or unsaturated
aliphatic chain having at least 2 carbons, and having at least one amine
subgroup; a linear or
branched, saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at
least one ether subgroup; a linear or branched, saturated or unsaturated
aliphatic chain
having at least 2 carbons, and having at least one halogen subgroup; a linear
or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons, and having
at least one
nitronium subgroup; wherein R5 is selected from the group consisting of. OH;
N02; NR';
OR'; wherein R' is selected from the group consisting of. a linear or
branched, saturated or
unsaturated aliphatic chain having at least one carbon; a linear or branched,
saturated or
unsaturated aliphatic chain having at least 2 carbons, and having at least one
hydroxyl
subgroup; a linear or branched, saturated or unsaturated aliphatic chain
having at least 2
carbons, and having at least one thiol subgroup; a linear or branched,
saturated or
unsaturated aliphatic chain having at least 2 carbons, wherein said aliphatic
chain terminates
with an aldehyde subgroup; a linear or branched, saturated or unsaturated
aliphatic chain
having at least 2 carbons, and having at least one ketone subgroup; a linear
or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons; wherein
said aliphatic
chain terminates with a carboxylic acid subgroup; a linear or branched,
saturated or
unsaturated aliphatic chain having at least 2 carbons, and having at least one
amide
subgroup; a linear or branched, saturated or unsaturated aliphatic chain
having at least 2
carbons, and having at least one acyl group; a linear or branched, saturated
or unsaturated
aliphatic chain having at least 2 carbons, and having at least one nitrogen
containing moiety
(e.g., nitro, nitrile, etc.); a linear or branched, saturated or unsaturated
aliphatic chain having
at least 2 carbons, and having at least one amine subgroup; a linear or
branched, saturated or
unsaturated aliphatic chain having at least 2 carbons, and having at least one
halogen
subgroup; a linear or branched, saturated or unsaturated aliphatic chain
having at least 2
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carbons, and having at least one nitronium subgroup; wherein R6 is selected
from the group
consisting of. Hyrdrogen; NO2; Cl; F; Br; I; SR'; and NR'2; wherein R' is
defined as above
in R5; wherein R7 is selected from the group consisting of. Hydrogen; a linear
or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons; and
wherein R8 is an
aliphatic cyclic group larger than benzene; wherein said larger than benzene
comprises any
chemical group containing 7 or more non-hydrogen atoms, and is an aryl or
aliphatic cyclic
group. In some embodiments, R' is any functional group that protects the
oxygen of R5
from metabolism in vivo, until the compound reaches its biological target
(e.g.,
mitochondria). In some embodiments, R' protecting group(s) is metabolized at
the target
site, converting R5 to a hydroxyl group.
In summary, a large number of benzodiazepine compounds and related compounds
are presented herein. Any one or more of these compounds can be used to treat
a variety of
dysregulatory disorders related to cellular death as described elsewhere
herein. The above-
described compounds can also be used in drug screening assays and other
diagnostic
methods.
V. Pharmaceutical compositions, formulations, and exemplary administration
routes and dosing considerations
Exemplary embodiments of various contemplated medicaments and pharmaceutical
compositions are provided below.
A. Preparing Medicaments
The compounds of the present invention are useful in the preparation of
medicaments to treat a variety of conditions associated with dysregulation of
cell death,
aberrant cell growth and hyperproliferation.
In addition, the compounds are also useful for preparing medicaments for
treating
other disorders wherein the effectiveness of the compounds are known or
predicted. Such
disorders include, but are not limited to, neurological (e.g., epilepsy) or
neuromuscular
disorders. The methods and techniques for preparing medicaments of a compound
are well-
known in the art. Exemplary pharmaceutical formulations and routes of delivery
are
described below.
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One of skill in the art will appreciate that any one or more of the compounds
described herein, including the many specific embodiments, are prepared by
applying
standard pharmaceutical manufacturing procedures. Such medicaments can be
delivered to
the subject by using delivery methods that are well-known in the
pharmaceutical arts.
B. Exemplary pharmaceutical compositions and formulation
In some embodiments of the present invention, the compositions are
administered
alone, while in some other embodiments, the compositions are preferably
present in a
pharmaceutical formulation comprising at least one active ingredient/agent
(e.g.,
benzodiazepine derivative), as defined above, together with a solid support or
alternatively,
together with one or more pharmaceutically acceptable carriers and optionally
other
therapeutic agents. Each carrier must be "acceptable" in the sense that it is
compatible with
the other ingredients of the formulation and not injurious to the subject.
Contemplated formulations include those suitable oral, rectal, nasal, topical
(including transdermal, buccal and sublingual), vaginal, parenteral (including
subcutaneous,
intramuscular, intravenous and intradermal) and pulmonary administration. In
some
embodiments, formulations are conveniently presented in unit dosage form and
are prepared
by any method known in the art of pharmacy. Such methods include the step of
bringing
into association the active ingredient with the carrier which constitutes one
or more
accessory ingredients. In general, the formulations are prepared by uniformly
and
intimately bringing into association (e.g., mixing) the active ingredient with
liquid carriers
or finely divided solid carriers or both, and then if necessary shaping the
product.
Formulations of the present invention suitable for oral administration may be
presented as discrete units such as capsules, cachets or tablets, wherein each
preferably
contains a predetermined amount of the active ingredient; as a powder or
granules; as a
solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-
water liquid
emulsion or a water-in-oil liquid emulsion. In other embodiments, the active
ingredient is
presented as a bolus, electuary, or paste, etc.
In some embodiments, tablets comprise at least one active ingredient and
optionally
one or more accessory agents/carriers are made by compressing or molding the
respective
agents. In preferred embodiments, compressed tablets are prepared by
compressing in a
suitable machine the active ingredient in a free-flowing form such as a powder
or granules,
optionally mixed with a binder (e.g., povidone, gelatin, hydroxypropylmethyl
cellulose),
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lubricant, inert diluent, preservative, disintegrant (e.g., sodium starch
glycolate, cross-linked
povidone, cross-linked sodium carboxymethyl cellulose)surface-active or
dispersing agent.
Molded tablets are made by molding in a suitable machine a mixture of the
powdered
compound (e.g., active ingredient) moistened with an inert liquid diluent.
Tablets may
optionally be coated or scored and may be formulated so as to provide slow or
controlled
release of the active ingredient therein using, for example,
hydroxypropylmethyl cellulose
in varying proportions to provide the desired release profile. Tablets may
optionally be
provided with an enteric coating, to provide release in parts of the gut other
than the
stomach.
Formulations suitable for topical administration in the mouth include lozenges
comprising the active ingredient in a flavored basis, usually sucrose and
acacia or
tragacanth; pastilles comprising the active ingredient in an inert basis such
as gelatin and
glycerin, or sucrose and acacia; and mouthwashes comprising the active
ingredient in a
suitable liquid carrier.
Pharmaceutical compositions for topical administration according to the
present
invention are optionally formulated as ointments, creams, suspensions,
lotions, powders,
solutions, pastes, gels, sprays, aerosols or oils. In alternatively
embodiments, topical
formulations comprise patches or dressings such as a bandage or adhesive
plasters
impregnated with active ingredient(s), and optionally one or more excipients
or diluents. In
preferred embodiments, the topical formulations include a compound(s) that
enhances
absorption or penetration of the active agent(s) through the skin or other
affected areas.
Examples of such dermal penetration enhancers include dimethylsulfoxide (DMSO)
and
related analogues.
If desired, the aqueous phase of a cream base includes, for example, at least
about
30% w/w of a polyhydric alcohol, i.e., an alcohol having two or more hydroxyl
groups such
as propylene glycol, butane- l,3-diol, mannitol, sorbitol, glycerol and
polyethylene glycol
and mixtures thereof.
In some embodiments, oily phase emulsions of this invention are constituted
from
known ingredients in an known manner. This phase typically comprises an lone
emulsifier
(otherwise known as an emulgent), it is also desirable in some embodiments for
this phase
to further comprises a mixture of at least one emulsifier with a fat or an oil
or with both a fat
and an oil.
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Preferably, a hydrophilic emulsifier is included together with a lipophilic
emulsifier
so as to act as a stabilizer. It some embodiments it is also preferable to
include both an oil
and a fat. Together, the emulsifier(s) with or without stabilizer(s) make up
the so-called
emulsifying wax, and the wax together with the oil and/or fat make up the so-
called
emulsifying ointment base which forms the oily dispersed phase of the cream
formulations.
Fmulgents and emulsion stabilizers suitable for use in the formulation of the
present
invention include TweenTM 60, SpanTM 80, cetostearyl alcohol, myristyl
alcohol, glyceryl
monostearate and sodium lauryl sulfate.
The choice of suitable oils or fats for the formulation is based on achieving
the
desired properties (e.g., cosmetic properties), since the solubility of the
active
compound/agent in most oils likely to be used in pharmaceutical emulsion
formulations is
very low. Thus creams should preferably be a non-greasy, non-staining and
washable
products with suitable consistency to avoid leakage from tubes or other
containers. Straight
or branched chain, mono- or dibasic alkyl esters such as di-isoadipate,
isocetyl stearate,
propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl
oleate, isopropyl
pahuitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain
esters known
as Crodamol CAP may be used, the last three being preferred esters. These may
be used
alone or in combination depending on the properties required. Alternatively,
high melting
point lipids such as white soft paraffin and/or liquid paraffin or other
mineral oils can be
used.
Formulations suitable for topical administration to the eye also include eye
drops
wherein the active ingredient is dissolved or suspended in a suitable carrier,
especially an
aqueous solvent for the agent.
Formulations for rectal administration may be presented as a suppository with
suitable base comprising, for example, cocoa butter or a salicylate.
Formulations suitable for vaginal administration may be presented as
pessaries,
creams, gels, pastes, foams or spray formulations containing in addition to
the agent, such
carriers as are known in the art to be appropriate.
Formulations suitable for nasal administration, wherein the carrier is a
solid, include
coarse powders having a particle size, for example, in the range of about 20
to about 500
microns which are administered in the manner in which snuff is taken, i.e., by
rapid
inhalation (e.g., forced) through the nasal passage from a container of the
powder held close
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up to the nose. Other suitable formulations wherein the carrier is a liquid
for administration
include, but are not limited to, nasal sprays, drops, or aerosols by
nebulizer, an include
aqueous or oily solutions of the agents.
Formulations suitable for parenteral administration include aqueous and non-
aqueous isotonic sterile injection solutions which may contain antioxidants,
buffers,
bacteriostats and solutes which render the formulation isotonic with the blood
of the
intended recipient; and aqueous and non-aqueous sterile suspensions which may
include
suspending agents and thickening agents, and liposomes or other
microparticulate systems
which are designed to target the compound to blood components or one or more
organs. In
some embodiments, the formulations are presented/formulated in unit-dose or
multi-dose
sealed containers, for example, ampoules and vials, and may be stored in a
freeze-dried
(lyophilized) condition requiring only the addition of the sterile liquid
carrier, for example
water for injections, immediately prior to use. Extemporaneous injection
solutions and
suspensions may be prepared from sterile powders, granules and tablets of the
kind
previously described.
Preferred unit dosage formulations are those containing a daily dose or unit,
daily
subdose, as herein above-recited, or an appropriate fraction thereof, of an
agent.
It should be understood that in addition to the ingredients particularly
mentioned
above, the formulations of this invention may include other agents
conventional in the art
having regard to the type of formulation in question, for example, those
suitable for oral
administration may include such further agents as sweeteners, thickeners and
flavoring
agents. It also is intended that the agents, compositions and methods of this
invention be
combined with other suitable compositions and therapies. Still other
formulations
optionally include food additives (suitable sweeteners, flavorings, colorings,
etc.),
phytonutrients (e.g., flax seed oil), minerals (e.g., Ca, Fe, K, etc.),
vitamins, and other
acceptable compositions (e.g., conjugated linoelic acid), extenders, and
stabilizers, etc.
C. Exemplary administration routes and dosing considerations
Various delivery systems are known and can be used to administer a therapeutic
agents (e.g., benzodiazepine derivatives) of the present invention, e.g.,
encapsulation in
liposomes, microparticles, microcapsules, receptor-mediated endocytosis, and
the like.
Methods of delivery include, but are not limited to, intra-arterial, intra-
muscular,
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intravenous, intranasal, and oral routes. In specific embodiments, it may be
desirable to
administer the pharmaceutical compositions of the invention locally to the
area in need of
treatment; this may be achieved by, for example, and not by way of limitation,
local
infusion during surgery, injection, or by means of a catheter.
The agents identified herein as effective for their intended purpose can be
administered to subjects or individuals susceptible to or at risk of
developing pathological
growth of target cells and condition correlated with this. When the agent is
administered to
a subject such as a mouse, a rat or a human patient, the agent can be added to
a
pharmaceutically acceptable carrier and systemically or topically administered
to the
subject. To determine patients that can be beneficially treated, a tissue
sample is removed
from the patient and the cells are assayed for sensitivity to the agent.
Therapeutic amounts are empirically determined and vary with the pathology
being
treated, the subject being treated and the efficacy and toxicity of the agent.
When delivered
to an animal, the method is useful to further confirm efficacy of the agent.
One example of
an animal model is MLR/MpJ-lpr/lpr ("MLR-lpr") (available from Jackson
Laboratories,
Bal Harbor, Maine). MLR-lpr mice develop systemic autoimmune disease.
Alternatively,
other animal models can be developed by inducing tumor growth, for example, by
subcutaneously inoculating nude mice with about 105 to about 109
hyperproliferative,
cancer or target cells as defined herein. When the tumor is established, the
compounds
described herein are administered, for example, by subcutaneous injection
around the
tumor. Tumor measurements to determine reduction of tumor size are made in two
dimensions using venier calipers twice a week. Other animal models may also be
employed
as appropriate. Such animal models for the above-described diseases and
conditions are
well-known in the art.
In some embodiments, in vivo administration is effected in one dose,
continuously or
intermittently throughout the course of treatment. Methods of determining the
most
effective means and dosage of administration are well known to those of skill
in the art and
vary with the composition used for therapy, the purpose of the therapy, the
target cell being
treated, and the subject being treated. Single or multiple administrations are
carried out
with the dose level and pattern being selected by the treating physician.
Suitable dosage formulations and methods of administering the agents are
readily
determined by those of skill in the art. Preferably, the compounds are
administered at about
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0.01 mg/kg to about 200 mg/kg, more preferably at about 0.1 mg/kg to about 100
mg/kg,
even more preferably at about 0.5 mg/kg to about 50 mg/kg. When the compounds
described herein are co-administered with another agent (e.g., as sensitizing
agents), the
effective amount may be less than when the agent is used alone.
The pharmaceutical compositions can be administered orally, intranasally,
parenterally or by inhalation therapy, and may take the form of tablets,
lozenges, granules,
capsules, pills, ampoules, suppositories or aerosol form. They may also take
the form of
suspensions, solutions and emulsions of the active ingredient in aqueous or
nonaqueous
diluents, syrups, granulates or powders. In addition to an agent of the
present invention, the
pharmaceutical compositions can also contain other pharmaceutically active
compounds or
a plurality of compounds of the invention.
More particularly, an agent of the present invention also referred to herein
as the
active ingredient, may be administered for therapy by any suitable route
including, but not
limited to, oral, rectal, nasal, topical (including, but not limited to,
transdermal, aerosol,
buccal and sublingual), vaginal, parental (including, but not limited to,
subcutaneous,
intramuscular, intravenous and intradermal) and pulmonary. It is also
appreciated that the
preferred route varies with the condition and age of the recipient, and the
disease being
treated.
Ideally, the agent should be administered to achieve peak concentrations of
the
active compound at sites of disease. This may be achieved, for example, by the
intravenous
injection of the agent, optionally in saline, or orally administered, for
example, as a tablet,
capsule or syrup containing the active ingredient.
Desirable blood levels of the agent may be maintained by a continuous infusion
to
provide a therapeutic amount of the active ingredient within disease tissue.
The use of
operative combinations is contemplated to provide therapeutic combinations
requiring a
lower total dosage of each component antiviral agent than may be required when
each
individual therapeutic compound or drug is used alone, thereby reducing
adverse effects.
D. Exemplary co-administration routes and dosing considerations
The present invention also includes methods involving co-administration of the
compounds described herein with one or more additional active agents. Indeed,
it is a
further aspect of this invention to provide methods for enhancing prior art
therapies and/or
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pharmaceutical compositions by co-administering a compound of this invention.
In co-
administration procedures, the agents may be administered concurrently or
sequentially. In
one embodiment, the compounds described herein are administered prior to the
other active
agent(s). The pharmaceutical formulations and modes of administration may be
any of
those described above. In addition, the two or more co-administered chemical
agents,
biological agents or radiation may each be administered using different modes
or different
formulations.
The agent or agents to be co-administered depends on the type of condition
being
treated. For example, when the condition being treated is cancer, the
additional agent can
be a chemotherapeutic agent or radiation. When the condition being treated is
an
autoimmune disorder, the additional agent can be an immunosuppressant or an
anti-
inflammatory agent. When the condition being treated is chronic inflammation,
the
additional agent can be an anti-inflammatory agent. The additional agents to
be co-
administered, such as anticancer, immunosuppressant, anti-inflammatory, and
can be any of
the well-known agents in the art, including, but not limited to, those that
are currently in
clinical use. The determination of appropriate type and dosage of radiation
treatment is also
within the skill in the art or can be determined with relative ease.
Treatment of the various conditions associated with abnormal apoptosis is
generally
limited by the following two major factors: (1) the development of drug
resistance and (2)
the toxicity of known therapeutic agents. In certain cancers, for example,
resistance to
chemicals and radiation therapy has been shown to be associated with
inhibition of
apoptosis. Some therapeutic agents have deleterious side effects, including
non-specific
lymphotoxicity, renal and bone marrow toxicity.
The methods described herein address both these problems. Drug resistance,
where
increasing dosages are required to achieve therapeutic benefit, is overcome by
co-
administering the compounds described herein with the known agent. The
compounds
described herein appear to sensitize target cells to known agents (and vice
versa) and,
accordingly, less of these agents are needed to achieve a therapeutic benefit.
The sensitizing function of the claimed compounds also addresses the problems
associated with toxic effects of known therapeutics. In instances where the
known agent is
toxic, it is desirable to limit the dosages administered in all cases, and
particularly in those
cases were drug resistance has increased the requisite dosage. When the
claimed
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compounds are co-administered with the known agent, they reduce the dosage
required
which, in turn, reduces the deleterious effects. Further, because the claimed
compounds are
themselves both effective and non-toxic in large doses, co-administration of
proportionally
more of these compounds than known toxic therapeutics will achieve the desired
effects
while minimizing toxic effects.
VI. Drug screens
In preferred embodiments of the present invention, the compounds of the
present
invention, and other potentially useful compounds, are screened for their
binding affinity to
the oligomycin sensitivity conferring protein (OSCP) portion of the
mitochondrial ATP
synthase complex. In particularly preferred embodiments, compounds are
selected for use
in the methods of the present invention by measuring their biding affinity to
recombinant
OSCP protein. A number of suitable screens for measuring the binding affinity
of drugs
and other small molecules to receptors are known in the art. In some
embodiments, binding
affinity screens are conducted in in vitro systems. In other embodiments,
these screens are
conducted in in vivo or ex vivo systems. While in some embodiments quantifying
the
intracellular level of ATP following administration of the compounds of the
present
invention provides an indication of the efficacy of the methods, preferred
embodiments of
the present invention do not require intracellular ATP or pH level
quantification.
Additional embodiments are directed to measuring levels (e.g., intracellular)
of
superoxide in cells and/or tissues to measure the effectiveness of particular
contemplated
methods and compounds of the present invention. In this regard, those skilled
in the art will
appreciate and be able to provide a number of assays and methods useful for
measuring
superoxide levels in cells and/or tissues.
In some embodiments, structure-based virtual screening methodologies are
contemplated for predicting the binding affinity of compounds of the present
invention with
OSCP.
Any suitable assay that allows for a measurement of the rate of binding or the
affinity of a benzodiazepine or other compound to the OSCP may be utilized.
Examples
include, but are not limited to, competition binding using Bz-423, surface
plasma resonace
(SPR) and radio-immunopreciptiation assays (Lowman et al., J. Biol.Chem.
266:10982
[1991]). Surface Plasmon Resonance techniques involve a surface coated with a
thin film
CA 02524394 2008-11-13
of a conductive metal, such as gold, silver, chrome or aluminum, in which
electromagnetic
waves, called Surface Plasmons, can be induced by a beam of light incident on
the metal
glass interface at a specific angle called the Surface Plasmon Resonance
angle. Modulation
of the refractive index of the interfacial region between the solution and the
metal surface
following binding of the captured macromolecules causes a change in the SPR
angle which
can either be measured directly or which causes the amount of light reflected
from the
underside of the metal surface to change. Such changes can be directly related
to the mass
and other optical properties of the molecules binding to the SPR device
surface. Several
biosensor systems based on such principles have been disclosed (See e.g., WO
90/05305).
There are also several commercially available SPR biosensors (e.g., BiaCoreTM,
Uppsala,
Sweden).
In some embodiments, copmpounds are screened in cell culture or in vivo (e.g.,
non-
human or human mammals) for their ability to modulate mitochondrial ATP
synthase
activity. Any suitable assay may be utilized, including, but not limited to,
cell proliferation
assays (Commercially available from, e.g., Promega, Madison, WI and
Stratagene, La Jolla,
CA) and cell based dimerization assays. (See e.g., Fuh et al., Science,
256:1677 [1992];
Colosi et al., J. Biol. Chem., 268:12617 [1993]). Additional assay formats
that find use
with the present invention include, but are not limited to, assays for
measuring cellular ATP
levels, and cellular superoxide levels.
The present invention also provides methods of modifying and derivatizing the
compositions of the present invention to increase desirable properties (e.g.,
binding affinity,
activity, and the like), or to minimize undesirable properties (e.g.,
nonspecific reactivity,
toxicity, and the like). The principles of chemical derivatization are well
understood. In
some embodiments, iterative design and chemical synthesis approaches are used
to produce
2S a library of derivatized child compounds from a parent compound. In other
embodiments,
rational design methods are used to predict and model in silico ligand-
receptor interactions
prior to confinning results by routine experimentation.
VII. Therapeutic Application
A. General Therapeutic Application
In particularly preferred embodiments, the compositions (e.g., benzodiazepine
derivatives)
of the present invention provide therapeutic benefits to patients suffering
from any one or
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more of a number of conditions (e.g., diseases characterized by dysregulation
of necrosis
and/or apoptosis processes in a cell or tissue, disease characterized by
aberrant cell growth
and/or hyperproliferation, etc.) by modulating (e.g., inhibiting or promoting)
the activity of
the mitochondrial ATP synthase (as referred to as mitochondrial F0F1 ATPase)
complexes
in affected cells or tissues. In further preferred embodiments, the
compositions of the
present invention are used to treat autoimmune/chronic inflammatory conditions
(e.g.,
psoriasis). In even further embodiments, the compositions of the present
invention are used
in conjunction with stenosis therapy to treat compromised (e.g., occluded)
vessels.
In particularly preferred embodiments, the compositions of the present
invention
inhibit the activity of mitochondrial ATP synthase complex by binding to a
specific subunit
of this multi-subunit protein complex. While the present invention is not
limited to any
particular mechanism, nor to any understanding of the action of the agents
being
administered, in some embodiments, the compositions of the present invention
bind to the
oligomycin sensitivity conferring protein (OSCP) portion of the mitochondrial
ATP
synthase complex. Likewise, it is further contemplated that when the
compositions of the
present invention bind to the OSCP the initial affect is overall inhibition of
the
mitochondrial ATP synthase complex, and that the downstream consequence of
binding is a
change in ATP or pH level and the production of reactive oxygen species (e.g.,
O2-). In still
other preferred embodiments, while the present invention is not limited to any
particular
mechanism, nor to any understanding of the action of the agents being
administered, it is
contemplated that the generation of free radicals ultimately results in cell
killing. In yet
other embodiments, while the present invention is not limited to any
particular mechanism,
nor to any understanding of the action of the agents being administered, it is
contemplated
that the inhibiting mitochondrial ATP synthase complex using the compositions
and
methods of the present invention provides therapeutically useful inhibition of
cell
proliferation.
Accordingly, preferred methods embodied in the present invention, provide
therapeutic benefits to patients by providing compounds of the present
invention that
modulate (e.g., inhibiting or promoting) the activity of the mitochondrial ATP
synthase
complexes in affected cells or tissues via binding to the oligomycin
sensitivity conferring
protein (OSCP) portion of the mitochondrial ATP synthase complex. Importantly,
by itself
the OSCP has no biological activity.
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Thus, in one-broad sense, preferred embodiments of the present invention are
directed to the discovery that many diseases characterized by dysregulation of
necrosis
and/or apoptosis processes in a cell or tissue, or diseases characterized by
aberrant cell
growth and/or hyperproliferation, etc., can be treated by modulating the
activity of the
mitochondrial ATP synthase complex including, but not limited to, by binding
to the
oligomycin sensitivity conferring protein (OSCP) component thereof. The
present
invention is not intended to be limited, however, to the practice of the
compositions and
methods explicitly described herein. Indeed, those skilled in the art will
appreciate that a
number of additional compounds not specifically recited herein (e.g., non-
benzodiazepine
derivatives) are suitable for use in the methods disclosed herein of
modulating the activity
of mitochondrial ATP synthase.
The present invention thus specifically contemplates that any number of
suitable
compounds presently known in the art, or developed later, can optionally find
use in the
methods of the present invention. For example, compounds including, but not
limited to,
oligomycin, ossamycin, cytovaricin, apoptolidin, bafilomyxcin, resveratrol,
piceatannol, and
dicyclohexylcarbodiimide (DCCD), and the like, find use in the methods of the
present
invention. The present invention is not intended, however, to be limited to
the methods or
compounds specified above. In one embodiment, that compounds potentially
useful in the
methods of the present invention may be selected from those suitable as
described in the
scientific literature. (See e.g., K.B. Wallace and A.A. Starkov, Annu. Rev.
Pharmacol.
Toxicol., 40:353-388 [2000]; A.R. Solomon et al., Proc. Nat. Acad. Sci.
U.S.A.,
97(26):14766-14771 [2000]).
In some embodiments, compounds potentially useful in methods of the present
invention are screened against the National Cancer Institute's (NCI-60) cancer
cell lines for
efficacy. (See e.g., A. Monks et al., J. Natl. Cancer Inst., 83:757-766
[1991]; and K.D.
Paull et al., J. Natl. Cancer Inst., 81:1088-1092 [1989]). Additional screens
suitable screens
(e.g., autoimmunity disease models, etc.) are within the skill in the art.
In one aspect, derivatives (e.g., pharmaceutically acceptable salts, analogs,
stereoisomers, and the like) of the exemplary compounds or other suitable
compounds are
also contemplated as being useful in the methods of the present invention.
In other preferred embodiments, the compositions of the present invention are
used
in conjunction with stenosis therapy to treat compromised (e.g., occluded)
vessels. In
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further embodiments, the compositions of the present invention are used in
conjunction with
stenosis therapy to treat compromised cardiac vessels.
Vessel stenosis is a condition that develops when a vessel (e.g., aortic
valve)
becomes narrowed. For example, aortic valve stenosis is a heart condition that
develops
when the valve between the lower left chamber (left ventricle) of the heart
and the major
blood vessel called the aorta becomes narrowed. This narrowing (e.g.,
stenosis) creates too
small a space for the blood to flow to the body. Normally the left ventricle
pumps oxygen-
rich blood to the body through the aorta, which branches into a system of
arteries
throughout the body. When the heart pumps, the 3 flaps, or leaflets, of the
aortic valve open
one way to allow blood to flow from the ventricle into the aorta. Between
heartbeats, the
flaps close to form a tight seal so that blood does not leak backward through
the valve. If the
aortic valve is damaged, it may become narrowed (stenosed) and blood flow may
be
reduced to organs in the body, including the heart itself. The long-term
outlook for people
with aortic valve stenosis is poor once symptoms develop. People with
untreated aortic
valve stenosis who develop symptoms of heart failure usually have a life
expectancy of 3
years or less.
Several types of treatment exist for treating compromised valves (e.g.,
balloon
dilation, ablation, atherectomy or laser treatment). One type of treatment for
compromised
cardiac valves is angioplasty. Angioplasty involves inserting a balloon-tipped
tube, or
catheter, into a narrow or blocked artery in an attempt to open it. By
inflating and deflating
the balloon several times, physicians usually are able to widen the artery.
A common limitation of angioplasty or valve expansion procedures is
restenosis.
Restenosis is the reclosure of a peripheral or coronary artery following
trauma to that artery
caused by efforts to open a stenosed portion of the artery, such as, for
example, by balloon
dilation, ablation, atherectomy or laser treatment of the artery. For these
angioplasty
procedures, restenosis occurs at a rate of about 20-50% depending on the
definition, vessel
location, lesion length and a number of other morphological and clinical
variables.
Restenosis is believed to be a natural healing reaction to the injury of the
arterial wall that is
caused by angioplasty procedures. The healing reaction begins with the
thrombotic
mechanism at the site of the injury. The final result of the complex steps of
the healing
process can be intimal hyperplasia, the uncontrolled migration and
proliferation of medial
89
CA 02524394 2008-11-13
smooth muscle cells, combined with their extracellular matrix production,
until the artery is
again stenosed or occluded.
In an attempt to prevent restenosis, metallic intravascular stents have been
permanently implanted in coronary or peripheral vessels. The stent is
typically inserted by
catheter into a vascular lumen told expanded into contact with the diseased
portion of the
arterial wall, thereby providing mechanical support for the lumen. However, it
has been
found that restenosis can still occur with such stents in place. Also, the
scent itself can cause
undesirable local thrombosis. To address the problem of thrombosis, persons
receiving
stents also receive extensive systemic treatment with anticoagulant and
antiplatelet drugs.
To address the restenosis problem, it has been proposed to provide stents
which are
seeded with endothelial cells (Dichek, D. A. et al Seeding of Intravascular
Stents With
Genetically Engineered Endothelial Cells; Circulation 1989; 80: 1347-1353). In
that
experiment, sheep endothelial cells that had undergone retrovirus-mediated
gene transfer for
either bacterial beta-galactosidase or human tissue-type plasminogen activator
were seeded
onto stainless steel stents and grown until the stents were covered. The cells
were therefore
able to be delivered to the vascular wall where they could provide therapeutic
proteins.
Other methods of providing therapeutic substances to the vascular wall by
means of stents
have also been proposed such as in international patent application WO
91/12779
"Intralurninal Drug Eluting Prosthesis" and international patent application
WO 90/13332
"Stent With Sustained Drug Delivery". In those applications, it is suggested
that antiplatelet
agents, anticoagulant agents, antimicrobial agents, anti-inflammatory agents,
antimetabolic
agents and other drugs could be supplied in stents to reduce the incidence of
restenosis.
Further, other vasoreactive agents such as nitric oxide releasing agents could
also be used.
An additional cause of restenosis is the over-proliferation of treated tissue.
In
preferred embodiments, the anti-proliferative properties of the present
invention inhibit
restenosis. Drug-eluting stents are well known in the art (see, e.g., U.S.
Patent No.:
5,697,967; U.S. Patent No.: 5,599,352; and U.S. Patent No.: 5,591,227 ),
In preferred embodiments, the compositions of the
present invention are eluted from drug-eluting stents in the treatment of
compromised (e.g.,
occluded) vessels. In further embodiments, the compositions of the present
invention are
eluted from drug-eluting stents in the treatment of compromised cardiac
vessels.
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WO 2005/004988 PCT/US2004/013455
Those skilled in the art of preparing pharmaceutical compounds and
formulations
will appreciate that when selecting optional compounds for use in the methods
disclosed
herein, that suitability considerations include, but are not limited to, the
toxicity, safety,
efficacy, availability, and cost of the particular compounds.
B. Autoimmune Disorder and Chronic Inflammatory Disorder Therapeutic
Application
Autoimmune disorders and chronic inflammatory disorders often result from
dysfunctional cellular proliferation regulation and/or cellular apoptosis
regulation.
Mitochondria perform a key role in the control and execution of cellular
apoptosis. The
mitochondrial permeability transition pore (MPTP) is a pore that spans the
inner and outer
mitochondrial membrandes and functions in the regulation of proapoptotic
particles.
Transient MPTP opening results in the release of cytochrolne c and the
apoptosis inducing
factor from the mitochondrial intermembrane space, resulting in cellular
apoptosis.
The oligomycin sensitivity conferring protein (OSCP) is a subunit of the FOFI
mitochondrial ATP synthase/ATPase and functions in the coupling of a proton
gradient
across the Fo sector of the enzyme in the mitochondrial membrane. In preferred
embodiments, compounds of the present invention binds the OSCP, increases
superoxide
and cytochrome c levels, increases cellular apoptosis, and inhibits cellular
proliferation.
The adenine nucleotide translocator (ANT) is a 30kDa protein that spans the
inner
mitochondrial membrane and is central to the mitochondrial permeability
transition pore
(MPTP). Thiol oxidizing or alkylating agents are powerful activators of the
MPTP that act
by modifying one or more of three unpaired cysteines in the matrix side of the
ANT. 4-(N-
(S-glutathionylacetyl)amino) phenylarsenoxide,
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CA 02524394 2008-11-13
(Co2
NH
O S
H
~~. N
`
HNH
O /
As OH
}H3N HO
C02
inhibits the ANT.
The compounds and methods of the present invention are useful in the treatment
of
autoimmune disorders and chronic inflammatory disorders. In such embodiments,
the
present invention provides a subject suffering from an autoimmune disorder
and/or a
chronic inflammatory disorder, and a composition comprising the following
formula(s):
O
'000O
As
I
0
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WO 2005/004988 PCT/US2004/013455
(CO2
NH
O S
H
N
HN~~\\\ H O
O
As OH
H3N HO
C02
R7
O
N
L
R6
R8
N
R1
Rq.
R2
R3
R5
wherein Rl, R2, R3 and R4 are selected from the group consisting of. hydrogen;
CH3; a
linear or branched, saturated or unsaturated aliphatic chain having at least 2
carbons; a
linear or branched, saturated or unsaturated aliphatic chain having at least 2
carbons, and
having at least one hydroxy subgroup; a linear or branched, saturated or
unsaturated
aliphatic chain having at least 2 carbons, and having at least one thiol
subgroup; a linear or
branched, saturated or unsaturated aliphatic chain having at least 2 carbons,
wherein said
aliphatic chain terminates with an aldehyde subgroup; a linear or branched,
saturated or
unsaturated aliphatic chain having at least 2 carbons, and having at least one
ketone
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WO 2005/004988 PCT/US2004/013455
subgroup; a linear or branched, saturated or unsaturated aliphatic chain
having at least 2
carbons; wherein said aliphatic chain terminates with a carboxylic acid
subgroup; a linear or
branched, saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at
least one amide subgroup; a linear or branched, saturated or unsaturated
aliphatic chain
having at least 2 carbons, and having at least one acyl group; a linear or
branched, saturated
or unsaturated aliphatic chain having at least 2 carbons, and having at least
one nitrogen
containing moiety (e.g.,nitro, nitrile, etc.); a linear or branched, saturated
or unsaturated
aliphatic chain having at least 2 carbons, and having at least one amine
subgroup; a linear or
branched, saturated or unsaturated aliphatic chain having at least 2 carbons,
and having at
least one ether subgroup; a linear or branched, saturated or unsaturated
aliphatic chain
having at least 2 carbons, and having at least one halogen subgroup; a linear
or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons, and having
at least one
nitronium subgroup; wherein R5 is selected from the group consisting of: OH;
N02; NR';
OR'; wherein R' is selected from the group consisting of. a linear or
branched, saturated or
unsaturated aliphatic chain having at least one carbon; a linear or branched,
saturated or
unsaturated aliphatic chain having at least 2 carbons, and having at least one
hydroxyl
subgroup; a linear or branched, saturated or unsaturated aliphatic chain
having at least 2
carbons, and having at least one thiol subgroup; a linear or branched,
saturated or
unsaturated aliphatic chain having at least 2 carbons, wherein said aliphatic
chain terminates
with an aldehyde subgroup; a linear or branched, saturated or unsaturated
aliphatic chain
having at least 2 carbons, and having at least one ketone subgroup; a linear
or branched,
saturated or unsaturated aliphatic chain having at least 2 carbons; wherein
said aliphatic
chain terminates with a carboxylic acid subgroup; a linear or branched,
saturated or
unsaturated aliphatic chain having at least 2 carbons, and having at least one
amide
subgroup; a linear or branched, saturated or unsaturated aliphatic chain
having at least 2
carbons, and having at least one acyl group; a linear or branched, saturated
or unsaturated
aliphatic chain having at least 2 carbons, and having at least one nitrogen
containing moiety
(e.g., nitro, nitrile, etc.); a linear or branched, saturated or unsaturated
aliphatic chain having
at least 2 carbons, and having at least one amine subgroup; a linear or
branched, saturated or
unsaturated aliphatic chain having at least 2 carbons, and having at least one
halogen
subgroup; a linear or branched, saturated or unsaturated aliphatic chain
having at least 2
carbons, and having at least one nitronium subgroup; wherein R6 is selected
from the group
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CA 02524394 2008-11-13
consisting of: Hyrdrogen; NO2; Cl; F; Br; 1; SR'; and NR'2; wherein R' is
defined as above
in R5; wherein R7 is selected from the group consisting of Hydrogen; a linear
or
branched, saturated or unsaturated aliphatic chain having at least 2 carbons;
and wherein R8
is an aliphatic cyclic group larger than benzene; wherein said larger than
benzene comprises
any chemical group containing 7 or more non-hydrogen atoms, and is an aryl or
aliphatic
cyclic group. In some embodiments, R' is any functional group that protects
the oxygen of
R5 from metabolism in vivo, until the compound reaches its biological target
(e.g.,
mitochondria). In some embodiments, R' protecting group(s) is metabolized at
the target
site, converting R5 to a hydroxyl group.
VIII. ATPase Inhibitors And Methods For Identifying Therapeutic Inhibitors
The present invention provides compounds that target the F1F,, ATPase. In
addition,
the present invention provides compounds that target the F1F,, ATPase as a
treatment for
autoimmune disorders, and in particular, compounds with low toxicity. The
present
invention further provides methods of identifying compounds that target the
F1F,, ATPase.
Additionally, the present invention provides therapeutic applications for
compounds
targeting the F1Fo ATPase.
A majority of ATP within eukaryotic cells is synthesized by the mitochondrial
F1Fo-
ATPase (see, e_g.,C.T. Gregory et at., J. Immunol., 139:313-318 [1987]; J.P.
Portanova et
al., Mol, Immunol., 32:117-135 [1987]; M.J. Shlomchik et al., Nat. Rev.
Immunol., 1:147-
153 [2001] ). Although the F1F,,
ATPase synthesizes and hydrolyzes ATP, during normal physiologic conditions,
the F1F0,-
ATPase only synthesizes ATP (see, e.g., Nagyvary J, et at., Biochem. Educ.
1999; 27:193-
99 ). The mitochondria] F1F,, ATPase is
composed of three major domains: F0, F1 and the peripheral stator. Fl is the
portion of the
enzyme that contains the catalytic sites and it is located in the matrix (see,
e.g., Boyer, PD,
Annu Rev Biochem.1997; 66:717-49 ). This
domain is highly conserved and has the subunit composition a303ybs. The
landmark X-ray
structure of bovine F1 revealed that 043 forms a hexagonal cylinder with they
subunit in the
center of the cylinder. F. is located within the inner mitochondrial membrane
and contains
a proton channel. Translocation of protons from the inner-membrane space into
the matrix
provides the energy to drive ATP synthesis. The peripheral stator is composed
of several
CA 02524394 2008-11-13
proteins that physically and functionally link F. with Fl. The stator
transmits
conformational changes from F,, into in the catalytic domain that regulate ATP
synthesis
(see, e.g., Cross RL, Biochim Biophys Acta 2000; 1458:270-75 ).
Mitochondrial F1Fo-ATPase inhibitors are invaluable tools for mechanistic
studies of
the FIFO-ATPase (see, e.g., James AM, et al., J Biomed Sci 2002; 9:475-87 ),
Because F1Fo-ATPase inhibitors are often
cytotoxic, they have been explored as drugs for cancer and other
hyperproliferative
disorders. Macrolides (e.g., oligomycin and apoptolidin) are non-competitive
inhibitors of
the F1Fo ATPase (see, e.g., Salomon AR, et al., PNAS 2000; 97:14766-71;
Salomon AR, et
al., Chem Biol 2001; 8:71-80 ). Macrolides
bind to F,, which blocks proton flow through the channel resulting in
inhibition of the F1F,,
ATPase. Macrolides are potent (e.g., the IC50 for oligomycin = 10 nM) and lead
to large
decreases in [ATP]. As such, macrolides have an unacceptably narrow
therapeutic index
and are highly toxic (e.g., the LD50 for oligomycin in rodents is two daily
doses at 0.5
mg/kg) (see, e.g., Kramar R. et al., Agents & Actions 1984, 15:660-63
Other inhibitors of F1Fo-ATPase include Bz-423, which binds
to the OSCP in F1 (as described elsewhere herein). Bz-423 has an K; -9 W.
In cells that are actively respiring (known as state 3 respiration),
inhibiting F1Fo
ATPase blocks respiration and places the mitochondria in a resting state
(known as state 4).
In state 4, the MRC is reduced relative to state 3, which favors reduction of
02 to 02- at
complex III (see, e.g., N. Zamzarni et al., J. Exp. Med., 181:1661-1672 [1995]
),
For example, treating cells with either oligomycin
or Bz-423 leads to a rise of intracellular 02 as a consequence of inhibiting
complex V. In
the case of oligomycin, supplementing cells with ATP protects against death
whereas
antioxidants do not, indicating that cell death results from the drop in ATP
(see, e.g., Zhang
JG, et al., Arch Biochem Biophys 2001; 393:87-96; McConkey DJ, et al., The ATP
switch
in apoptosis. In: Nieminen La, ed. Mitochondria in pathogenesis. New York:
Plenum,
2001:265-77 ). Bz-423-induced cell
death is blocked by antioxidants and is not affected by supplementing cells
with ATP,
indicating that Bz-423 engages an ROS-dependent death response (see, e.g.,
N.B. Blatt, et
al., J. Clin. Invest., 2002, 110, 1123 ). As
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WO 2005/004988 PCT/US2004/013455
such, F1Fo-ATPase inhibitors are either toxic (e.g., oligomycin) or
therapeutic (e.g., Bz-
423).
The present invention provides a method of distinguishing toxic F1F,, ATPase
inhibitors from therapeutic F1Fo ATPase inhibitors. F1F,, ATPase inhibitors
with
therapeutic potential (e.g., Bz-423) present a novel mode of inhibition.
Specifically, F1F0,-
ATPase inhibitors with beneficial properties like Bz-423 are uncompetitive
inhibitors that
only bind enzyme-substrate complexes at high substrate concentration and do
not alter the
kcat/K,,, ratio. This knowledge forms the basis to identify and distinguish
F1Fo ATPase
inhibitors with therapeutic potential from toxic compounds.
The present invention provides compounds that target the F1Fo ATPase as an
autoimmune disorder treatment. In particular, the present invention provides
methods of
identifying compounds that target the F1Fo-ATPase while not altering the
kcat/K,,, ratio.
Additionally, the present invention provides therapeutic applications for
compounds
targeting the F1F,,ATPase.
A. ATPase Inhibiting Compounds
The present invention provides compounds that inhibit the F1Fo ATPase. In some
embodiments, the compounds do not bind free F1Fo ATPase, but rather bind to an
F1F,,
ATPase-substrate complex. The compounds show maximum activity at high
substrate
concentration and minimal activity (e.g., F1Fo-ATPase inhibiting) at low
substrate
concentration. In preferred embodiments, the compounds do not alter the
kcat/Km ratio of the
F1Fo ATPase. The properties of the F1Fo ATPase inhibitors of the present
invention are in
contrast with oligomycin, which is a F1Fo ATPase inhibitor that is acutely
toxic and lethal.
Oligomycin is a noncompetitive inhibitor, which binds to both free F1Fo ATPase
and F1Fo
ATPase-substrate complexes and alters the kcat/Km ratio.
The compounds of the present invention that inhibit F1Fo-ATPase while not
altering the
kcat/Km ratio, in some embodiments, have the structure described elsewhere
herein.
However, compounds of other structures that are identified as therapeutic
inhibitors by the
methods of the present invention are also encompassed by the present
invention.
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B. Identifying ATPase Inhibitors
The present invention provides methods of identifying (e.g., screening)
compounds
useful in treating autoimmune disorders. The present invention is not limited
to a particular
type compound. In preferred embodiments, compounds of the present invention
include, but
are not limited to, pharmaceutical compositions, small molecules, antibodies,
large molecules,
synthetic molecules, synthetic polypeptides, synthetic polynucleotides,
synthetic nucleic acids,
aptamers, polypeptides, nucleic acids, and polynucleotides. The present
invention is not
limited to a particular method of identifying compounds useful in treating
autoimmune
disorders. In preferred embodiments, compounds useful in treating autoimmune
disorders are
identified as possessing an ability to inhibit an F1F,, ATPase while not
altering the kcat/Km ratio.
C. Therapeutic Applications With F1F0,-ATPase Inhibitors
The present invention provides methods for treating disorders (e.g.,
neurodegenerative
diseases, Alzheimers, ischemia reprofusion injury, neuromotor disorders, non-
Hodgkin's
lymphoma, lymphocytic leukemia, cutaneous T cell leukemia, an autoimmune
disorder, cancer,
solid tumors, lymphomas, and leukemias). The present invention is not limited
to a particular
form of treatment. In preferred embodiments, treatment includes, but is not
limited to,
symptom amelioration, symptom prevention, disorder prevention, and disorder
amelioration.
The present invention provides methods of treating autoimmune disorders
applicable within in
vivo, in vitro, and/or ex vivo settings.
In some embodiments, the present invention treats autoinunune disorders
through
inhibiting of target cells. The present invention is not limited to a
particular form of cell
inhibition. In preferred embodiments, cell inhibition includes, but is not
limited to, cell growth
prevention, cell proliferation prevention, and cell death. In preferred
embodiments, inhibition
of a target cell is accomplished through contacting a target cell with an F1Fo
ATPase inhibitor
of the present invention. In further embodiments, target cell inhibition is
accomplished
through targeting of the F1Fo ATPase with an F1Fo ATPase inhibitor of the
present invention.
The present invention is not limited to a particular F1Fo ATPase inhibitor. In
preferred
embodiments, the F1Fo ATPase inhibitor possesses the ability to inhibit an
F1Fo ATPase while
not altering the kcat/Km ratio. In further preferred embodiments, the F1Fo
ATPase inhibitor is
Bz-423 or other compounds described herein.
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The present invention further provides methods for selectively inhibiting the
pathology of target cells in a subject in need of therapy. The present
invention is not limited
to a particular method of inhibition target cell pathology. In preferred
embodiments, target
cell pathology is inhibited through administration of an effective amount of a
compound of
the invention. The present invention is not limited to a particular compound.
In preferred
embodiments, the compound is an F1F0,-ATPase inhibitor. In further preferred
embodiments, the compound inhibits the F1F,,-ATPase while not altering the
ratio.
EXAMPLES
The following examples are provided to demonstrate and further illustrate
certain
preferred embodiments of the present invention and are not to be construed as
limiting the
scope thereof.
Example 1
Preparation of Compounds
The benzodiazepine compounds are prepared using either solid-phase or soluble-
phase combinatorial
synthetic methods as well as on an individual basis from well-established
techniques. See for example, C.J
Boojamra et al. in "Solid-Phase Synthesis of 1, 4-Benzodiazepine-2,5-diones.
Library Preparation and
Demonstration of Synthesis Generality", J. Org. Chem. (1997) 62, pp. 1240-
1256; Bunin et at in "The
Combinatorial Synthesis and Chemical and Biological Evaluation of a 1,4-
Benzodiazepine Library," Proc. Natl.
Acad. Sci. U.S.A. (1994) 91, pp.4708-4712; Stevens et al. in "Non Nucleic Acid
Inhibitors of Protein: DNA
Interactions Identified through Combinatorial Chemistry", J. Am. Chem. Soc.
(1996) 118, pp. 10650-1065 1;
Gordon et al. in "Applications of Combinatorial Technologies to Drug
Discovery. 2. Combinatorial Organic
Synthesis, Library Screening Strategies, and Future Directions," J. Med. Chem.
(1994) 37, pp. 1385-1401; U.S.
Patent No. 4,110,337 issued on August 29, 1978 and U.S. Patent No. 4,076,823
issued on February 28, 1978. For
illustration, the following general methodologies are provided.
Preparation of 1,4-benzodiazepine-2-one compounds
Improved solid-phase synthetic methods for the preparation of a variety of 1,4-
benzodiazepine-2-one derivatives with very high overall yields have been
reported in the
literature. (See e.g., Bunin and Ellman, J. Am. Chem, Soc., 114:10997-10998
[1992]).
Using these improved methods, the 1,4-benzodiazepine-2-ones is constructed on
a solid
support from three separate components: 2-aminobenzophenones, a-amino acids,
and
(optionally) alkylating agents.
Preferred 2-aminobenzophenones include the substituted 2-aminobenzophenones,
for example, the halo-, hydroxy-, and halo-hydroxy-substituted 2-
aminobenzophenones,
such as 4-halo-4'-hydroxy-2-aminobenzophenones. A preferred substituted 2-
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aminobenzophenone is 4-chloro-4'-hydroxy-2-aminobenzophenone. Preferred a-
amino
acids include the 20 common naturally occurring a-amino acids as well as a-
amino acid
mimicking structures, such as homophenylalanine, homotyrosine, and thyroxine.
Alkylating agents include both activated and inactivated electrophiles, of
which a
wide variety are well known in the art. Preferred alkylating agents include
the activated
electrophiles p-bromobenzyl bromide and t-butyl-bromoacetate.
In the first step of such a synthesis, the 2-aminobenzophenone derivative is
attached
to a solid support, such as a polystyrene solid support, through either a
hydroxy or
carboxylic acid functional group using well known methods and employing an
acid-
cleavable linker, such as the commercially available [4-
(hydroxyrnethyl)phenoxy] acetic
acid, to yield the supported 2-aminobenzophenone. (See e.g., Sheppard and
Williams, Intl.
J. Peptide Protein Res., 20:451-454 [1982]). The 2-amino group of the
aminobenzophenone
is preferably protected prior to reaction with the linking reagent, for
example, by reaction
with FMOC-Cl (9-fluorenylmethyl chloroformate) to yield the protected amino
group 2'-
NHFMOC.
In the second step, the protected 2-amino group is deprotected (for example,
the -
NHFMOC group may be deprotected by treatment with piperidine in
dimethylformamide
(DMF)), and the unprotected 2-aminobenzophenone is then coupled via an amide
linkage to
an a-amino acid (the amino group of which has itself been protected, for
example, as an -
NHFMOC group) to yield the intermediate. Standard activation methods used for
general
solid-phase peptide synthesis are used (such as the use of carbodiimides and
hydroxybentzotriazole or pentafluorophenyl active esters) to facilitate
coupling. However,
a preferred activation method employs treatment of the 2-aminobenzophenone
with a
methylene chloride solution of the of a-N-FMOC-amino acid fluoride in the
presence of the
acid scavenger 4-methyl-2,6-di-tert-butylpyridine yields complete coupling via
an amide
linkage. This preferred coupling method has been found to be effective even
for unreactive
aminobenzophenone derivatives, yielding essentially complete coupling for
derivatives
possessing both 4-chloro and 3-carboxy deactivating substituents.
In the third step, the protected amino group (which originated with the amino
acid)
is first deprotected (e.g., -NHFMOC may be converted to -NH2 with piperidine
in DMF),
and the deprotected Bz-423s reacted with acid, for example, 5% acetic acid in
DMF at
60 C, to yield the supported 1,4-benzodiazepine derivative. Complete
cyclization has been
100
CA 02524394 2008-11-13
reported using this method for a variety of 2-aminobenzophenone derivatives
with widely
differing steric and electronic properties.
In an optional fourth step, the 1,4-benzodiazepine derivative is alkylated, by
reaction
with a suitable allcylatiog agent and a base, to yield the supported fully
derivatized 1,4-
benzodiazepine. Standard alkylation methods, for example, an excess of a
strong base such
as LDA (lithium diisopropylamide) or NaH, is used; however, such methods may
result in
undesired deprotonation of other acidic functionalities and over-allcylation.
Preferred bases,
which may prevent over-alkylation of the benzodiazepine derivatives (for
example, those
with ester and carbamate functionalities), are those which are basic enough to
completely
deprotonate the anilide functional group, but not basic enough to deprotonate
amide,
carbamate or ester functional groups. An example of such a base is lithiated 5-
(phenyhnethyl)-2-oxaxolidinone, which is reacted with the 1,4-benzodiazepine
in
tetrahydrofuran (THF) at -78 C. Following deprotonation, a suitable alkylating
agent, as
described above, is added.
In the final step, the fully derivatized 1,4-benzodiazepine is cleaved from
the solid
support. This is achieved (along with concomitant removal of acid-labile
protecting
groups), for example, by exposure to a suitable acid, such as a mixture of
trifluoroacetic
acid, water, and dimethylsulfide (85:5:10, by volume). Alternatively, the
above
benzodiazepines is prepared in soluble phase. The synthetic methodology was
outlined by
Gordon et al., J. Med. Chem., 37:1386-1401 [1994]),
Briefly, the methodology comprises trans-imidating an amino acid resin with
appropriately substituted 2-aminobenzophenone imines to form resin-bound
imines. These
imines are cyclized and tethered by procedures similar to those in solid-phase
synthesis
described above. The general purity of benzodiazepines prepared using the
above
methodology is about 90% or higher.
Preparation of 1,4-benzodiazepine-2,5-diones
A general method for the solid-phase synthesis of 1,4-benzodiazepine-2,5-
diones has
been reported in detail by C.J. Boojamra et al., J. Org. Chem., 62:1240-1256
[1996]). This
method is used to prepare the compounds of the present invention.
AMerrifield resin, for example, a (chloromethyl)polystyrene is derivatized by
allcylation with 4-hydroxy-2,6-dimethoxybenzaldehyde sodium to provide resin-
bound
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aldehyde. An a-amino ester is then attached to the derivatized support by
reductive
arnination using NaBH(OAc)3 in 1% acetic acid in DMF. This reductive amination
results
in the formation of a resin-bound secondary amine.
The secondary amine is acylated with a wide variety of unprotected anthranilic
acids
result in support-bound tertiary amides. Acyla.tion is best achieved by
performing the
coupling reaction in the presence of a carbodiimide and the hydrochloride salt
of a tertiary
amine. One good coupling agent is 1-ethyl-8-[8-(dimethylamino)propyl]
carbodiimide
hydrochloride. The reaction is typically performed in the presence of
anhydrous 1-methyl-
2-pyrrolidinone. The coupling procedure is typically repeated once more to
ensure
complete acylation.
Cyclization of the acyl derivative is accomplished through base-catalyzed
lactamation through the formation of an anilide anion which would react with
an alkylhalide
for simultaneous introduction of the substituent at the 1-position on the
nitrogen of the
heterocyclic ring of the benzodiazepine. The lithium salt of acetanilide is a
good base to-
catalyze the reaction. Thus, the Bz-423s reacted with lithium acetanilide in
DMF/THF (1:1)
for 30 hours followed by reaction with appropriate alkylating agent provides
the fully
derivatized support-bound benzodiazepine. The compounds are cleaved from the
support in
good yield and high purity by using TFA/DMS/H20 (90:5:5).
Some examples of the a-amino ester starting materials, alkylating agents, and
anthranilic acid derivates that
are used in the present invention are listed by Boojamra et al. in "Solid-
Phase Synthesis of 1, 4-Benzodiazepine-2,5-
diones. Library Preparation and Demonstration of Synthesis Generality", J.
Org. Chem. (1997) 62, pp. 1240-1256;
Additional reagents are readily determined and either are commercially
obtained or readily prepared by one of
ordinary skill in the art to arrive at the novel substituents disclosed in the
present invention.
For example, from Boojamra, supra, one realizes that: allylating agents
provide the
R1 substituents; a-amino ester starting materials provide the R2 substituents,
and anthranilic
acids provide the R4 substituents. By employing these starting materials that
are
appropriately substituted, one arrives at the desired 1.4-benzodiazepine-2,5-
dione. The R3
substituent is obtained by appropriately substituting the amine of the a-
aminoester starting
material. If steric crowding becomes a problem, the R3 substituent is attached
through
conventional methods after the 1,4-benzodiazepine-2,S-dione is isolated.
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Example 2
Chirality
It should be recognized that many of the benzodiazepines of the present
invention
exist as optical isomers due to chirality wherein the stereocenter is
introduced by the a-
amino acid and its ester starting materials. The above-described general
procedure
preserves the chirality of the a-amino acid or ester starting materials. In
many cases, such
preservation of chirality is desirable. However, when the desired optical
isomer of the a-
amino acid or ester starting material is unavailable or expensive, a racemic
mixture is
produced which is separated into the corresponding optical isomers and the
desired
benzodiazepine enantiomer is isolated.
For example, in the case of the 2,5-dione compounds, Boojamra, supra,
discloses
that complete racernization is accomplished by preequilibrating the
hydrochloride salt of the
enantiomerically pure a-amino ester starting material with 0.3 equivalents of
i-Pr2EtN and
the resin-bound aldehyde for 6 hours before the addition of NaBH(OAc)3. The
rest of the
above-described synthetic procedure remains the same. Similar steps are
employed, if
needed, in the case of the 1,4-benzodiazepine-2-dione compounds as well.
Methods to prepare individual benzodiazepines are well-known in the art. (See
e.g.,
U.S. 3,415,814; 3,384,635; and 3,261,828 ). By
selecting the appropriately substituted starting materials in any of the above-
described
methods, the benzodiazepines of this invention are prepared with relative
ease.
Example 3
Reagents
Bz-423 is synthesized as described above. FK506 is obtained from Fujisawa
(Osaka, Japan). N-benzoylcarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD) is
obtained
from Enzyme Systems (Livermore, CA). Dihydroethidium (DHE) and 3,3'-
dihexyloxacarbocyanine iodide (DiOC6(3)) are obtained from Molecular Probes
(Eugene,
OR). FAM-VAD-fink is obtained from Intergen (Purchase, NJ). Manganese(M)meso-
tetrakis(4-benzoic acid)porphyrin (MnTBAP) is purchased from Alexis
Biochemicals (San
Diego, CA). Benzodiazepines is synthesized as described (See, B.A. Bunin et
al., Proc.
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Natl. Acad. Sci. U.S.A., 91:4708-4712 [1994]). Other reagents were obtained
from Sigma
(St. Louis, MO).
Example 4
Animals and drug delivery
Female NZB/W mice (Jackson Labs, Bar Harbor, ME) are randomly distributed into
treatment and control groups. Control mice receive vehicle (50 L aqueous
DMSO) and
treatment mice receive Bz-423 dissolved in vehicle (60 mg/kg) through
intraperitoneal
injections. Peripheral blood is obtained from the tail veins for the
preparation of serum.
Samples of the spleen and kidney are preserved in either 10% buffered-formalin
or by
freezing in OCT. An additional section of spleen from each animal is reserved
for the
preparation of single cell suspensions.
Example 5
Primary splenocytes, cell lines, and culture conditions
Primary splenocytes are obtained from 6 month old mice by mechanical
disruption
of spleens with isotonic lysis of red blood cells. B cell-rich fractions are
prepared by
negative selection using magnetic cell sorting with CD4, CD8a and CD1 lb
coated
microbeads (Miltenyi Biotec, Auburn, California). The Ramos line is purchased
from the
ATCC (Monassis, Georgia). Cells are maintained in RPMI supplemented with 10%
heat-
inactivated fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin (100
gg/ml) and
L-glutamine (290 g/ml). Media for primary cells also contains 2-
mercaptoethanol (50
M). All in vivo studies are performed with 0.5% DMSO and 2% FBS. In vitro
experiments are conducted in media containing 2% FBS. Organic compounds are
dissolved
in media containing 0.5% DMSO.
Example 6
Histology
Formalin-fixed kidney sections were stained with hematoxylin and eosin (H&E)
and
glomerular immune-complex deposition is detected by direct immunofluorescence
using
frozen tissue stained with FITC-conjugated goat anti-mouse IgG (Southern
Biotechnology,
Birmingham, AL). Sections are analyzed in a blinded fashion for nephritis and
IgG
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deposition using a 0-4+ scale. The degree of lymphoid hyperplasia is scored on
a 0-4+
scale using spleen sections stained with H&E. To identify B cells, sections
are stained with
biotinylated-anti-B220 (Pharmingen; 1 .tg/mL) followed by streptavidin-Alexa
594
(Molecular Probes; 5 g/mL). Frozen spleen sections are analyzed for TUNEL
positive
cells using an In situ Cell Death Detection kit (Roche) and are evaluated
using a 0-4+ scale.
Example 7
TUNEL staining
Frozen spleen sections are analyzed using an In. situ Cell Death Detection kit
(Roche
Molecular Biochemicals, Indianapolis, IN). Sections are blindly evaluated and
assigned a
score (0-4+) on, the basis of the amount of TUNEL-positive staining. B cells
are identified
by staining with biotinylated-anti-B220 (Pharmingen, San Diego, CA; 1 g/mL, 1
h, 22 C)
followed by streptavidin-Alexa 594 (Molecular Probes, Eugene, Oregon; 5
[Ig/mL, 1 h, 22
C)
Example 8
Flow cytometric analysis of spleen cells from treated animals
Surface markers are detected (15 in, 4 C) with fluorescent-conjugated anti-
Thy 1.2
(Pharmingen, 1 gg/mL) and/or anti-B220 (Pharmingen, 1 gg/mL). To detect outer-
membrane phosphatidyl serine, cells are incubated with FITC-conjugated Annexin
V and
propidium iodide (PI) according to manufacturer protocols (Roche Molecular
Biochemicals). Detection of TUNEL-positive cells by flow cytometry uses the
APO-
BRDU kit (Pharmingen). Superoxide and MPT are assessed by incubation of cells
for 30 in
at 27 degrees C with 10 gM dihydroethidium and 2 M 3,3'-
dihexyloxacarbocyanine iodide
(DIOC6(3)) (Molecular Probes). Prodidium idodie is used to determine viability
and DNA
content. Samples are analyzed on a FACSCalibur flow cytometer (Becton
Dickinson, San
Diego, CA).
Example 9
B cell stimulation
Ramos cells are activated with soluble goat Fab2 anti-human IgM (Southern
Biotechnology Associates, 1 g/ml) and/or purified anti-human CD40
(Pharmingen, clone
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5C3, 2.5 gg/ml). Mouse B cells are activated with affinity purified goat anti-
mouse IgM
(ICN, Aurora, Ohio; 20 g/ml) immobilized in culture wells, and/or soluble
purified anti-
mouse CD40 (Pharmingen, clone HM40-3, 2.5 gg/ml). LPS is used at 10 gg/ml. Bz-
423 is
added to cultures immediately after stimuli are applied. Inhibitors are added
30 in prior to
Bz-423.
Example 10
Statistical analysis
Statistical analysis is conducted using the SPSS software package. Statistical
significance is assessed using the Mann-Whitney U test and correlation between
variables is
assessed by two-way ANOVA. All p-values reported are one-tailed and data are
presented
as mean SEM.
Example 11
Detection of cell death and hypodiploid DNA
Cell viability is assessed by staining with propidium iodide (PI, 1 g/ml,).
PI
fluorescence is measured using a FACScalibur flow cytometer (Becton Dickinson,
San
Diego, CA). Measurement of hypodiploid DNA is conducted after incubating cells
in
DNA-labeling solution (50 gg/mL of PI in PBS containing 0.2% Triton and 10
g.g/mL
RNAse A) overnight at 4 degrees C. The data is analyzed using the CellQuest
software
excluding aggregates.
Example 12
Detection of 02 -, iV., and caspase activation
To detect 02-, cells are incubated with DHE (10 M) for 30 min at 37 C and
are
analyzed by flow cytometry to measure ethidium fluorescence. Flow analysis of
mitochondrial transmembrane potential (Wm) is conducted by labeling cells with
DiOC6(3)
(20 nM) for 15 min at 37 degrees C. A positive control for disruption of Wm is
established
using carbonyl cyanide m-chlorophenylhydrazone (CCCP, 50 M). Caspase
activation
assays are performed with FAM-VAD-fluoromethylketone. Processing of the
substrate is
evaluated by flow cytometry.
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Example 13
Subcellular fractionation and cytochrome c detection
Ramos cells (250 x 106 cells/sample) are treated with Bz-423 (10 M) or
vehicle for
1 to 5 h. Cells are pelleted, re-suspended in buffer (68 mM sucrose, 220 mM
mannitol, 10
mM HEPES-NaOH, pH 7.4, 10 mM ICI, 1 mM EDTA, 1 mM EGTA, 10 g/mL leupeptin,
gg/mL aprotinin, 1 mM PMSF), incubated on ice for 10 min, and homogenized. The
homogenate is centrifuged twice for 5 min at 4 C (800g) to pellet nuclei and
debris and for
min at 4 C (16,000g) to pellet mitochondria. The supernatant is concentrated,
electrophoresed on 12% SDS-PAGE gels, and transferred to Hybond ECL membranes
10 (Amersham, Piscataway, NJ). After blocking (PBS containing 5% dried milk
and 0.1%
Tween), the membranes are probed with an anti-cytochrome c monoclonal antibody
(Pharmingen, San Diego, CA; 2 g/mL) followed by an anti-mouse horseradish
peroxidase-
conjugated secondary with detection by chemiluminescence (Amersham).
15 Example 14
ROS production in isolated mitochondria
Male Long Evans rats are starved overnight and sacrificed by decapitation.
Liver
samples are homogenized in ice cold buffer A (250 mM sucrose, 10 mM Tris, 0.1
mM
EGTA, pH 7.4), and nuclei and cellular debris are pelleted (10 min, 830g, 4
C).
Mitochondria are collected by centrifugation (10 min, 15,000g, 4 C), and the
supernatant is
collected as the S 15 fraction. The mitochondrial pellet is washed three times
with buffer B
(250 mM sucrose, 10 mM Tris, pH 7.4), and re-suspended in buffer B at 20-30
mg/mL.
Mitochondria are diluted (0.5 mg/mL) in buffer C (200 mM sucrose, 10 mM Tris,
pH 7.4, 1
mm KH2PO4, 10 M EGTA, 2.5 M rotenone, 5 mM succinate) containing 2',7'-
dichlorodihydrofluorescin diacetate (DCFH-DA, 1 M). For state 3 measurements,
ADP (2
mM) is included in the buffer, and prior to the addition of Bz-423,
mitochondria are allowed
to charge for 2 min. To induce state 4, oligomycin (10 M) is added to buffer
C. The
oxidation of DCFH to 2',7'-dichlorofluorescein (DCF) is monitored at 37 C
with a
spectrofluorimeter (X,x: 503 nm; Xem: 522 nm). To detect effects on 02 and
delta Wm,
mitochondria are incubated for 15 min at 37 C in buffer C with vehicle, Bz-
423, or CCCP
containing DHE (5 M) or DIOC6(3) (20 nM), and aliquots are removed for
analysis by
fluorescence microscopy.
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Example 15
Flow cytometric analysis of splenocytes
Splenocytes are prepared by mechanical disruption and red blood cells removed
by
isotonic lysis. Cells are stained at 4 C with fluorescent-conjugated anti-Thy
1.2
(Pharmingen; I .tg/n1L) and/or anti-B220 (Phanningen; 1 g/mL) for 15 nun. To
detect
outer-membrane phosphatidyl serine, cells are incubated with FITC-conjugated
Annexin V
and PI (Roche Molecular Biochemicals, Indianapolis, IN; 1 g/mL).
Example 16
In vivo determination of ROS
Spleens are removed from 4-mo old NZB/W mice treated with Bz-423 or vehicle
and frozen in OCT. ROS production is measured using manganese(II)3,3,9-
diaminobenzidine as described in B.D. Kerver et al. (See, E.D. Kerver et al.,
Histochem. J.,
29:229-237 [1997).
Example 17
IgG titers, BUN, and proteinuria
Anti-DNA and IgG titers are determined by ELISA as described in P.C. Swanson
et
al. (See, P.C. Swanson et al., Biochemistry, 35:1624-1.633 [1996]). Serum BUN
is
measured by the University of Michigan Hospital's clinical laboratory.
Proteinuria is
monitored using ChemStrip 6 (Boehringer Mannheim).
Example 18
Benzodiazepine studies
Benzodiazepine studies on animals are described in U.S. Patent Publication
No.:
20010016583, published August 23, 2001.
Example 19
Mediators of Bz-423 induced apoptosis.
To characterize the death mechanism engaged by Bz-423, intracellular ROS,
AT,,n,
cytochrome c release, caspase activation, and DNA fragmentation were measured
over time
(the results presented are for B cells but do characterize the response in
many different cell
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types). The first event detected after exposure to Bz-423 is an increase in
the fraction of
cells that stain with dihyroethedium (DHE), a redox-sensitive agent that
reacts specifically
with 02--
Levels of 02 diminished after an early maximum at 1 hour and then increased
again
after 4 hours of continued treatment. This bimodal pattern pointed to a
cellular mechanism
limiting 02 and suggested that the "early" and "late" 02 maxima resulted from
different
processes.
Collapse of A'm was detected using DiOC6(3), a mitochondria-selective
potentiometric probe. The gradient change began after the early 02_ response
and was
observed in >90% of cells by 5 hours.
Cytochrome c release from mitochondria, a key step enabling caspase
activation,
was studied by immunoblotting cytosolic fractions. Levels of cytosolic
cytochrome c above
amounts in cells treated with vehicle were detected by 5 hours. This release
was coincident
with the disruption of AT, and together, these results were consistent with
opening of the
PT pore. Indeed, the late increase in O2 tracked with the A`P,,, collapse and
the release of
cytochrome c, suggesting that the secondary rise in 02 resulted from these
processes.
Caspase activation was measured by processing of the pan-caspase sensitive
fluorescent substrate FAM-VAD-fmk. Caspase activation tracked with AT, whereas
the
appearance of hypodiploid DNA was slightly delayed with respect to caspase
activation.
Collectively, these results indicated that Bz-423 induces a mitochondrial-
dependent
apoptotic pathway.
Example 20
Bz-423 directly targets mitochondria.
Since the early 02- preceded other cellular events, it was possible that this
ROS had
a regulatory role. In non-phagocytic cells, redox enzymes, along with the MRC,
are the
primary sources of ROS. Inhibitors of these systems were assayed for an
ability to regulate
Bz-423-induced 02 in order to determine the basis for this response. Of these
reagents,
only NaN3, which acts primarily on cytochrome c oxidase (complex IV of the
mitochondrial
respiratory chain, MRC), and micromolar amounts of FK506, which block the
formation of
02_ by the ubiquinol-cytochrome c reductase component of MRC complex III,
modulated
Bz-423. These findings suggested that mitochondria are the source of Bz-423-
induced 02-
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and that a component of the MRC is involved in the response. Although the
inhibition by
FK506 may result from binding to either calcineurin or FK506-binding proteins,
natural
products that bind tightly to these proteins (rapamycin and cyclosporin A,
respectively) did
not diminish the Bz-423 02- response.
02" production by Bz-423 may result from binding to a protein within
mitochondria
or a target in another compartment that signals mitochondria to generate ROS.
To
distinguish between these alternatives, isolated rat liver mitochondria were
assayed for ROS
production by monitoring the oxidation of 2',7'-dichlorodihydrofluorescin
diacetate to of
2',7'-dichlorofluorescin in the presence and absence of Bz-423. In this assay,
the rate of
DCF production increased after a lag period during which endogenous reducing
equivalents
were consumed and the acetate moieties on the probe were hydrolyzed to yield
2',7'-
dichlorodihydrofluorescin, the redox-active species. Under aerobic conditions
supporting
state 3 respiration, both antimycin A, which generates 02- by inhibiting
ubiquinol-
cytochrome c reductase, and Bz-423 increased the rate of ROS production nearly
two-fold
after the induction phase, based on comparing the slopes of each curve to
control. Swelling
was not observed, demonstrating that Bz-423 does not directly target the MPT
pore.
Neither Bz-423 nor antimycin A generated substantial ROS in the subcellular S
15 fraction
(cytosol and microsomes), and Bz-423 does not stimulate ROS if mitochondria
are in state
4, even though antimycin A is active under these conditions. Together, these
experiments
demonstrate that mitochondria contain a molecular target for Bz-423, and state
3 respiration
is required for the 02" response.
Example 21
Bz-423-induced ROS comes from mitochondria
MRC complexes I and III are the primary sources of ROS within mitochondria.
Evidence presented above suggests that Bz-423-induced ROS comes from
mitochondria.
To test this hypothesis, MRC function was knocked out the resulting cells were
examined
for ROS in response to Bz-423. Complexes I-IV in the MRC are partially encoded
by
mitochondrial DNA (mtDNA). Culturing cells over extended periods of time in
the
presence of ethidium bromide removed mtDNA, suggesting that mtDNA encoded
proteins
are not produced and electron transport along the MRC does not occur (cells
devoid of
mtDNA and associated proteins are often termed p cells). Because ethidium
bromide is
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toxic to Ramos cells, these experiments were conducted with Namalwa B cells,
another
mature B cell line. Treating Namalwa p0 cells with Bz-423 did not result in an
ROS
response, as was observed in both Ramos and Namalwa p+ cells.
Since the early ROS is critical to Bz-423 induced apoptosis, results detected
with the
Namalwa p0 cells would seemingly predict that these cells would be protected
from the
toxic effects of Bz-423. However, after 6 hours, the MPT was triggered and
Namalwa p0
cells underwent apoptosis in response to Bz-423. In p+ cells, proton pumping
by the MRC
maintained the mitochondrial gradient AT,,,. Since a functional MRC is not
present in p0
cells, 4`1',,, is supported by complex V (theFiFo-ATPase) functioning as an
ATPase (deletion
of subunits 6 and b in p0 cells abolishes the synthase activity of this
enzyme). In this case,
inhibition of complex V ATPase would cause collapse of the gradient and
subsequent cell
death.
Example 22
Bz-423 targets the mitochondrial F1Fo-ATPase
Oligomycin, a macrolide natural product that binds to the mitochondrial F1FO-
ATPase, induces a state 3 to 4 transition and generates 02- like Bz-423. Based
on these
similarities, it is possible that the F1F0-ATPase is also the molecular target
for Bz-423. To
test this hypothesis, the effect of Bz-423 on ATPase activity in sub-
mitochondrial particles
(SMPs) was examined. Indeed, Bz-423 inhibited the mitochondrial ATPase
activity of
bovine SMPs with an ED 50 ca. 5 M.
>40 derivatives of Bz-423 were developed to determine the elements on this
novel
agent required for biological activity. Assessing these compounds in whole
cell apoptosis
assays revealed that a hydroxyl group at the C'4 position and an aromatic ring
roughly the
size of the napthyl moiety were useful. The potency of these analogues in cell
based assays
correlated with the ED50 values in ATPase inhibition experiments using SMPs.
These
observations indicated that the mitochondrial ATPase is the molecular target
of Bz-423. At
concentrations where these derivatives are cytotoxic (80 M), other
benzodiazepines and
PBR ligands (e.g., PK1 1195 and 4-chlorodiazepam) do not significantly inhibit
mitochondrial ATPase activity, suggesting that the molecular target of Bz-423
is distinct
from the molecular target(s) of these other compounds.
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Example 23
Bz-423 binds to the OSCP
As part an early group of mechanistic studies of Bz-423, a biotinylated
analogue was
synthesized by replacing the N-methyl group with a hexylaminolinker to which
biotin was
covalently attached (this modification did not alter the activity of Bz-423).
This molecule
was used to probe a display library of human breast cancer cDNAs (Invitrogen)
that are
expressed as fusion proteins on the tip of T7 phage. Following the screening
methods
described by Austin and co-workers using biotinylated version of KF506 to
identify new
FK506 binding proteins, the OSCP component of the mitochondrial F1F0-ATPase
was
identified as a binding protein for Bz-423 (Figure 1).
To determine if Bz-423 indeed binds to the OSCP and the affinity of the
interaction,
human OSCP was overexpressed in E. coli. Titrating a solution of Bz-423 into
the OSCP
resulted in quenching of the intrinsic protein fluorescence and afforded a Kd
of 200 J 40 nM
(Figure 2). The binding of several Bz-423 analogues was also measured and it
was found
that their affinity for the OSCP paralleled their potency in both whole cell
cytotoxicity
assays as well as ATPase inhibition experiments using SMP. These data provided
cogent
evidence that Bz-423 binds to the OSCP on the mitochondrial ATPase. Bz-423 is
the only
known inhibitor of the ATPase that functions through binding to the OSCP.
Since the
OSCP does not contain the ATP binding site and it does not comprise the proton
channel, it
is possible that Bz-423 functions by altering the molecular motions of the
ATPase motor.
Example 24
RNAi knockouts of the OSCP protect against Bz-423 induced cell death
To complement the chemical and biochemical target identification and
validation
studies described above, experiments were conducted to knockout the OSCP in
whole cells.
In vitro, removing the OSCP from the ATPase abolishes synthase function
without altering
the hydrolytic activity of the enzyme. In yeast, OSCP knockouts are not
lethal; in these
cells, hydrolysis of ATP provides the chemical potential to support AY..
thereby maintaining
mitochondrial integrity. Since yeast OSCP has limited sequence homology to the
mammalian protein (-30%), these experiments were conducted in cell lines from
human
origin.
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Since the OSCP is nuclear encoded, RNA interference (RNAi), a technique that
can
achieve post-transcriptional gene silencing, was employed to knockout this
protein. For
these experiments, HEII 293 cells were transfected with each of three
chemically
synthesized small interfering RNA molecules (siRNA) specific for the OSCP
sequence
using oligofectamine. These cells are transfected in a highly efficient (90%)
manner by
oligofectamine. OSCP expression was analyzed by immunoblot at 24h, 48h, 72h
and 96h
after transfection. The maximum silencing of OSCP expression (64%) occurred at
72h after
transfection (Figure 3). OSCP siRNA transfected HEIR 293 cells had a reduced
Bz-ROS
and apoptosis in response to Bz-423 relative to cells transfected with a
scrambled sequence
control siRNA. These results indicated that siRNA is effective at reducing
OSCP and
suggested that Bz-423 mediated cell death signaling involves the OSCP.
Example 25
Effect of Bz-423 on cellular proliferation
Like most 1,4-benzodiazepines, Bz-423 binds strongly to bovine serum albumin
(BSA), which reduces the effective concentration of drug free in solution. For
example, in
tissue culture media containing 10% (v/v) fetal bovine serum (FBS), ca. 99% of
the drug is
bound to BSA. Therefore, cell culture cytotoxicity assays are conducted in
media with 2%
FBS to reduce binding to BSA and increase the free [Bz-423]. Under these
conditions, the
dose response-curve is quite sharp such that there is a limited concentration
range at which
Bz-423 is only partly effective. Since some benzodiazepines are known to have
anti-
proliferative properties, the effect of Bz-423 at concentrations < ED50 were
carefully
analyzed and observed that in addition to inducing apoptosis, Bz-423 prevented
cell growth
after 3 d in culture. In these low serum conditions, the cytotoxic and anti-
proliferative
effects overlapped making it difficult to study each effect independently.
However, by
increasing the [BSA] or increasing FBS to 10%, the dose-response curve
flattened (and the
cytotoxicity ED50 increased) and Bz-423 induced cytotoxicicty could be clearly
distinguished from effects on proliferation. At lower amounts of drug (e.g.,
10-15 M), Bz-
423 had minimal cytotoxicity whereas at concentrations > 20 M only apoptosis
was
observed (the death pathway described above including a bimodal ROS response,
and was
also observed in media containing 10% FBS). While higher amounts of drug may
also
block proliferation, it caused apoptosis well before the effects on
proliferation could be
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observed. Dose response curves were similar in experiments where BSA was added
to
media containing 2% FBS to simulate media containing 10% FBS, which
demonstrated that
antiproliferation and cytotoxicity were not affected by other constituents of
serum.
To confirm the decrease in cell number relative to control cells after 3 d of
treatment
is due to decreased proliferation and not cell death balanced by
proliferation, in addition to
cell counting, cell divisions were studied. PKH-67 is a fluorescent probe that
binds
irreversibly to cell membranes and upon cell division is partitioned equally
between the
daughter cells, making it possible to quantify cell division by flow
cytometry. Ramos cells
stained with PKH67 and treated with Bz-423 had fewer cell divisions at sub-
cytotoxic
concentrations which confirmed that the decrease in cell number was due to
anti-
proliferative affects and not cell death. To determine if Bz-423 induced anti-
proliferation
was specific to Ramos cells, cell counting and cell cycle experiments were
done in other B
cell lines and cell lines derived from solid tumors. As seen in Table 3, the
effects on
blocking proliferation were not unique to lymphoid cells which suggested a
target, common
to multiple tissue types, mediated the block in proliferation.
Table 3. ED50 ( M) for antiproliferation of cells treated for 72 h in media
with 10% FBS. Cells for study
included Ramos cells and clones transfected to overexpress Bcl-2 and Bcl-xL,
ovarian cells with null p53
(SKOV3); neuroblastoma cell lines (IMR-32, Lan-1, SHEP-1); and malignant B
cell lines.
Ramos Bcl-2 Bcl-XL SKOV3 IMR-32 Lan-1 SHEP-1 CA46 Raji
10.7 11.9 13.7 18.2 18.0 13.7 15.9 13.4 12.9
Example 26
Gene profiling cells treated with Bz-423.
Gene profiling experiments were conducted to probe the mechanism by which Bz-
423
blocks cellular proliferation. In studies using cyclohexamide as an inhibitor
of protein
synthesis, it was found that Bz-423 -induced cell death did not depend on new
protein
synthesis. Therefore, changes in gene expression were more likely relevant
only to the
mechanism of anti-proliferation. To increase the likelihood of detecting
changes involved
in signal-response coupling rather than down-stream effects, cells were
profiled that were
treated with Bz-423 for 3 h. This is the point just after the ROS early
maximum, but before
other cellular changes occur, including opening of the mitochondria
permeability pore.
The discovery of the pro-apoptotic, cytotoxic and growth inhibitory properties
of
Bz-423 against pathogenic cell types identified the potential for this class
of agents to be
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CA 02524394 2008-11-13
therapeutic against autoimmune diseases, cancers and other neoplastic
diseases. Further
experimental evidence from an analysis of the changes in gene expression
induced by this
agent expanded the mechanistic understanding of this compound's action and
added to the
collection of therapeutic effects it modulates.
In vitro testing with Ramos cells to determine the changes in gene expression
(at the
level of mRNA) induced by Bz-423 was performed by culturing cells at a density
of
500,000 cells per ml. Solvent control (DMSO, final concentration 0.1 % VV]),
Bz-423, or
Bz-OMe (10 M) was added to cells. After 4 h, cells were harvested and RNA
prepared
using Trizol Reagent (#15596-018, Life Technologies, Rockville, MD) and the
RNeasyTM
Maxi Kit (# 75162, Qiagen, Valencia, CA) according to manufacturers protocols.
Single
stranded cDNA was synthesized by reverse transcription using poly (A) RNA
present in the
starting total RNA sample. Single stranded eDNA was converted into double
stranded
eDNA and then in vitro transcription carried out in the presence of
biotinylated UTP and
CTP to produce biotin-labeled cRNA. cRNA was fragmented in the presence of
Mg2+, and
hybridized to the human genome U1 33A Genechip array (Affymetrix).
Hybridization
results were quantified using a GeneArray scanner and analysis carried out
according to the
instructions provided by Affymetrix.
Expression profiling using RNA isolated from cells treated with Bz-423, Bz-
OMe,
or vehicle control was done with the HGU133A Affymetrix gene chip, which
represents
about 22,000 human genes. Using criteria that includep<0.01, 16 genes are
expressed 8-
fold or more over control cells. As expected based on the molecular target of
Bz-423, many
of these genes were involved in glycolysis.
The data were analyzed to detect genes changes Bz treatment according to the
criteria that the log-transformed mean signal changed at least four-fold in
treated compared
to vehicle control samples and that the coefficient of variance for control
values (n=4) was
less than 10%. These genes represent targets that may mediate therapeutic
responses.
The gene expression results for Bz-423 and Bz-OMe each provide a unique
fingerprint of information. The structure of Bz-OMe is as follows:
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WO 2005/004988 PCT/US2004/013455
CH3
O
C1 N
/ \
OCH3
Expression of some genes change similarly after exposure to both Bz-423 and Bz-
OMe. Thus, the genes that are commonly regulated between the two compounds are
particularly relevant for understanding gene regulation through a more general
class of
compounds. Figure 4 presents data showing gene expression profiles of cells
treated by Bz-
423 and Bz-OMe.
Example 27
Effect of Bz-423 on ODC levels and activity
To determine whether ODC activity and polyamine metabolism is affected by Bz-
423, as suggested by RNA profiling data, ODC activity in cells treated with Bz-
423 was
directly measured in comparison with a vehicle control. In these experiments,
the
conversion of omithine to putrescine was quantified using 3H-ornithine. For
comparisons,
control cells were treated with vehicle control or difluoromethyl ornithine
(DFMO), a potent
inhibitor of ornithine decarboxylase (like Bz-423, DFMO is a potent anti-
proliferative
agent). As seen in Figure 3, treating cells for 4 h with Bz-423 significantly
reduced ODC
activity in a dose-dependant fashion, which is consistent with among other
things, an
incrrease in antizyme 1, as suggested by RNA profiling. The reduction in ODC
activity was
paralleled by a decrease in ODC protein levels measured under the same
conditions.
As described above, Bz-423 induced apoptosis was signaled by an ROS response
that arose from MRC complex III as a result of the state 3 to 4 transition. It
was next sought
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to determine if the ROS response, critical for apoptosis, also mediated these
effects on
ODC. If the ROS was required for the decrease in ODC activity, it would
likewise be
implicated as potentially part of the anti-proliferative response to Bz-423.
To test this,
Ramos cells were treated with Bz-423, DFMO, or vehicle control for 4 h. In
parallel, a
second group of cells was pre-incubated with MnTBAP to limit the ROS and then
cultured
with Bz-423, DFMO, and vehicle control. MnTBAP significantly reversed
inhibition of
ODC by Bz-423.
Collectively these data suggested the possible interpretation that high [Bz-
423]
(e.g.>10 M) generate sufficient amounts of ROS that could not be detoxified
by cellular
anti-oxidants, and resulted in apoptosis within 18 h. Lower [Bz-423] induced a
proportionally smaller ROS response that was insufficient to trigger
apoptosis. In this case,
however, the ROS may be capable of inhibiting ODC or otherwise blocking
cellular
proliferation.
Consistent with this hypothesis, a compound in which the phenolic hydroxyl is
replaced by Cl (designated Bz-Cl) was minimally cytotoxic (activity decreased
by ca 80%
compared to Bz-423) and generated a small ROS response in cells, while also
binding less
tightly to the OSCP (Kd 5 M). This compound also inhibited ODC activity
(Figure 3),
as predicted by the above hypothesis. Given the proposed role and nature of Bz-
423 induced
ROS in mediating growth arrest, Bz-Cl was tested against the panel of cells in
Table 2 and
found that after 3 d it reduced proliferation to a similar extent as Bz-423,
with comparable
ED50 values. These results demonstrated that the antiproliferative effects of
these
compounds could be obtained using chemical analogues of Bz-423 that block
proliferation
without inducing apoptosis.
Example 28
Structure Activity Studies of Novel Cytotoxic Benzodiazepines
Based on these properties of Bz-423, a range of Bz-423 derivatives were
synthesizedto probe structural elements of this novel compound important for
binding and
activity. Replacing the N-methyl group or chlorine with a hydrogen had little
effect on
lymphotoxic activity against immortalized Ramos B cells or Jurkat T cells in
culture.
Similarly, both enantiomers of Bz-423 were equipotent, which indicates that
the interaction
between Bz-423 and its molecular target involves two-point binding. In
contrast to these
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WO 2005/004988 PCT/US2004/013455
data, removing a naphthalalanine (see Table 1). The present invention is not
limited to a
particular mechanism, and an understanding of a mechanism is not necessary to
practice the
present invention, nonetheless, it is contemplated that moiety or replacing
the phenolic
hydroxyl group with hydrogen abolished all cytotoxic activity (Table 1). Based
on these
observations changes to the C'3 and C'4 positions were investigated. Replacing
1 -naphthol
with 2-naphtho has little effect on cell killing. Similarly, replacing the
napthylalanine with
other hydrophobic groups of comparable size had little effect on cytotoxic
properties of Bz-
423. By contrast, quinolines 7-9 were each less potent than Bz-423. The
present invention
is not limited to a particular mechanism, and an understanding of a mechanism
is not
necessary to practice the present invention, nonetheless, it is contemplated
that theses data
suggest a preference for a hydrophobic substituent within the binding site for
Bz-423.
Smaller C3 substituents were only somewhat less potent than Bz-423 whereas
compounds
with aromatic groups containing oxygen were significantly less cytotoxic.
These data
clearly indicate that a bulky hydrophobic aromatic substituent is useful for
optimal activity.
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Table 1. Potency of Bz-423 derivatives. Cell death was assessed by culturing
Ramos B cells in the presence of each compound in a dose-response fashion.
Cell
viability was measured after 24 h propidium iodide exclusion using flow
cytometry.
In this assay, the EC50 for PK1 1195, diazepam, and 4-Cl-diazepam is > 80 M.
CD o ~ 1
- naphthalAla 1 >80
\ 10 12
- phenol 2 >80
F
3 5 \ F 11 10
4 4 12 6
7
\ / \ / \ 13 7
6 4 14 35
OCF3
~ ~ 7 11 , I
25
8 12 OH
N
ICQ
i-\~ 9 15
'Each EC50 value was determined twice in triplicate and has an error of 5%.
Placing a methyl group ortho to the hydroxyl (16) does not alter the activity
of Bz-
423 whereas moving the hydroxyl to the C'4 (17) position decreased potency 2-
fold (Table
2). By contrast, replacing the hydroxyl with chlorine or azide, or methylating
the phenol
5 effectively abolishes the cytotoxic activity of Bz-423. The present
invention is not limited
to a particular mechanism, and an understanding of the mechanism is not
necessary to
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WO 2005/004988 PCT/US2004/013455
practice the present invention, nonetheless, it is contemplated that these
data indicate that a
hydroxyl group positioned at the C'4 carbon is required for optimal activity,
possibly by
making a critical contact upon target binding. However, molecules possessing a
phenolic
substructure can also act as alternate electron carriers within the MRC. Such
agents accept
an electron from MRC enzymes and transfer it back to the chain at point of
higher reducing
potential. This type of `redox cycling' consumes endogenous reducing
equivalents (e.g.,
glutathione) along with pyrimidine nucleotides and results in cell death. To
distinguish
between these alternatives, it was determined whether Bz-423 redox cycles in
the presence
of sub-mitochondrial particles using standard NADH and NAD(P)H oxidation
assays.
Unlike the positive controls (doxorubicin and menadione), Bz-423 does not lead
to substrate
oxidation which strongly suggests that it does not redox cycle. The present
invention is not
limited to a particular mechanism, and an understanding of the mechanism is
not necessary
to practice the present invention, nonetheless, it is contemplated that
collectively, the data
indicate that the decreased activity of compounds 18-20 results from removing
an
interaction that mediates binding of Bz-423 to its target protein.
Table 2. Potency of Bz-423 derivatives. Cell death was assessed as
described in Table 1
&
H
OH CI N3 OCH3
Compound 16 17 18 19 20
EC50 3 6 >80 >80 >80
Cells rapidly produce 02_ in response to Bz-423 and blocking this signal
(e.g., by
inhibiting ubiquinol cytochrome c reductase, which is the enzyme that produces
02 in
response to Bz-423) prevents apoptosis. To determine if the Bz-423 derivatives
kill cells in
manner analogous to Bz-423 (presumably as a result of binding to a common
molecular
target), the ability of FK506 was examined, micromolar amounts of which
effectively
inhibit ubiquinol cytochrome c reductase, to protect against cell death.
Inhibition by FK506
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CA 02524394 2008-11-13..
60%) was only observed for 3-6, 12, 13, 16, and 17, which are the compounds
with
hydrophobic C3 side chains larger than benzene. Cell death induced by each of
these
compounds (including Bz-423) was also inhibited (to = 60%) by pre-treating
cells with
either 18, 19, or 20 (at > 40 M). Compounds 18, 19, and 20 had no effect on
blocking the
cytotoxic activity (inhibition of = 20%) of the other benzodiazepines listed
in Table 2. The
present invention is not limited to a particular mechanism, and an
understanding of the
mechanism is not necessary to practice the present invention, nonetheless, it
is contemplated
that these data strongly suggest that Bz-423 along with 3-6, 12, 13, 16, and
17 bind the
same site within the target protein and induce apoptosis through a common
mechanism.
The other compounds do not bind at this site and induce a death response
through a
different pathway.
Example 29
Measuring ATPase activity
Mitochondria were isolated from the hearts of freshly slaughtered cattle as
previously described (see, e.g., Graham, J.M., Subcellular Fractionation and
Isolation of
Organelles: Isolation of Mitochondria fi=orn Tissues and Cells by Differential
Centrifugation., in Current Protocols in Cell Biology. 1999, John Wiley &
Sons, Inc: New
York. p. 3.3.3-3.3.4 ). All buffers contained
2-mercaptoethanol (5 mM). Submitochondrial particles (SMPs) were prepared by
sonication of beef heart mitochondria according to Walker et al (see, e.g.,
Walker, J.E., et
al., Methods Enzymol, 1995. 260: p. 163-90 )
except that each portion of mitochondria] suspension was sonicated three times
for
40 seconds, with an interval of two minutes between sonications, using a
Misonix sonicator
3000 with a 0.5-in titanium probe at energy setting 8.5, Mitochondrial F1Fo-
ATPase
activity was measured by coupling the production of ADP to the oxidation of
NADH via the
pyruvate kinase and lactate dehydrogenase reaction, and then monitoring the
rate of NADH
oxidation spectrophotometrically at 340 run at 30 C (see, e.g., McEnery, M.W.
et al., J Biol
Chem, 1986. 261(4): p. 1745-52; Harris, D.A., Spectrophotonretric Assays, in
Spectrophotonmetry and Spectrofluorimetry, D.A. Harris, Bashford, C.L.,
Editor. 1987, IRL
Press ). The reaction mixture (0.25
mL final volume) contained: Tris-HCl (100 mM), pH 8.0, ATP (0-2 mM), MgC12 (2
mM),
KCl (50 mM), EDTA (0.2 mM), NADH (0.2 mM), phosphoenolpyruvate (1 nrM),
pyruvate
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CA 02524394 2008-11-13
kinase (0.5 II), and lactate dehydrogenase (0.5 U). Each sample contained SMPs
(7 g) or
purified F1-ATPase (0.29 g) that had been pre-incubated (5 min at 30 C) with
various
concentrations of Bz-423 (or vehicle control).
Although the invention has been described in connection with
specific preferred embodiments, it should be understood that the invention as
claimed
should not be unduly limited to such specific embodiments. Indeed, various
modifications
of the described modes for carrying out the invention that are obvious to
those skilled in the
relevant fields are intended to be within the scope of the following claims.
122
