U.S. patent application number 09/745858 was filed with the patent office on 2001-09-06 for ortho-diphenol compounds, methods and pharmaceutical compositions for inhibiting parp.
This patent application is currently assigned to GUILFORD PHARMACEUTICALS, INC.. Invention is credited to Li, Jia-He, Serdyuk, Larisa E., Zhang, Jie.
Application Number | 20010020013 09/745858 |
Document ID | / |
Family ID | 22840037 |
Filed Date | 2001-09-06 |
United States Patent
Application |
20010020013 |
Kind Code |
A1 |
Zhang, Jie ; et al. |
September 6, 2001 |
Ortho-diphenol compounds, methods and pharmaceutical compositions
for inhibiting parp
Abstract
This invention relates to compounds, pharmaceutical
compositions, and methods of using compounds of the formula; 1 A is
O or S; R is C.sub.1-C.sub.10 straight or branched chain alkyl,
C.sub.2-C.sub.10 straight or branched chain alkenyl,
C.sub.2-C.sub.10 straight or branched chain alkynyl, aryl,
heteroaryl, carbocycle, or heterocycle; D is a bond, or a
C.sub.1-C.sub.3 straight or branched chain alkyl, C.sub.2-C.sub.3
straight or branched chain alkenyl, C.sub.2-C.sub.3 straight or
branched chain alkynyl, wherein any of the carbon atoms of said
alkyl, alkenyl, or alkynyl of D are optionally replaced with
oxygen, nitrogen, or sulfur; and X is aryl, heteroaryl, carbocycle,
or heterocycle.
Inventors: |
Zhang, Jie; (Ellicott City,
MD) ; Serdyuk, Larisa E.; (Baltimore, MD) ;
Li, Jia-He; (Cockeysville, MD) |
Correspondence
Address: |
NIXON & VANDERHYE P.C.
8th Floor
1100 North Glebe Rd.
Arlington
VA
22201-4714
US
|
Assignee: |
GUILFORD PHARMACEUTICALS,
INC.
|
Family ID: |
22840037 |
Appl. No.: |
09/745858 |
Filed: |
December 26, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09745858 |
Dec 26, 2000 |
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09224294 |
Dec 31, 1998 |
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6201020 |
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Current U.S.
Class: |
514/150 ;
514/423; 514/427; 514/456; 514/539; 534/848 |
Current CPC
Class: |
A61P 13/12 20180101;
A61P 21/04 20180101; A61P 25/04 20180101; A61P 35/00 20180101; A61P
17/02 20180101; C07C 327/26 20130101; A61P 43/00 20180101; A61P
9/10 20180101; A61P 9/02 20180101; A61P 21/02 20180101; A61P 25/28
20180101; A61P 1/04 20180101; A61P 25/16 20180101; A61P 7/08
20180101; A61P 19/10 20180101; C07C 69/017 20130101; A61P 25/02
20180101; A61P 19/02 20180101; A61P 37/00 20180101; A61P 25/14
20180101; A61P 21/00 20180101; A61P 3/10 20180101; A61P 25/18
20180101; A61P 27/02 20180101; A61P 25/00 20180101; A61P 37/04
20180101; A61P 31/18 20180101 |
Class at
Publication: |
514/150 ;
514/423; 514/427; 514/539; 514/456; 534/848 |
International
Class: |
A61K 031/655; A61K
031/40 |
Claims
We claim:
1. A compound of formula (I): 36or a pharmaceutically acceptable
salt, hydrate, ester, solvate, prodrug, metabolite, stereoisomer,
or mixtures thereof, wherein A is O or S; R is C.sub.1-C.sub.10
straight or branched chain alkyl, C.sub.2-C.sub.10 straight or
branched chain alkenyl, C.sub.2-C.sub.10 straight or branched chain
alkynyl, aryl, heteroaryl, carbocycle, or heterocycle; D is a bond,
or a C.sub.1-C.sub.3 straight or branched chain alkyl,
C.sub.2-C.sub.3 straight or branched chain alkenyl, C.sub.2-C.sub.3
straight or branched chain alkynyl, wherein any of the carbon atoms
of said alkyl, alkenyl, or alkynyl of D are optionally replaced
with oxygen, nitrogen, or sulfur; and X is aryl, heteroaryl,
carbocycle, or heterocycle; wherein said alkyl, alkenyl, alkynyl,
aryl, heteroaryl, carbocycle, or heterocycle of R, D, or X is
optionally substituted with one or more substituents selected from
hydroxy, halo, haloalkyl, thiocarbonyl, alkoxy, alkenoxy,
alkylaryloxy, aryloxy, arylalkyloxy, cyano, nitro, amino, imino,
alkylamino, arylamino, arylazo, arylthio, aminoalkyl,
sulfhydryl,,thioalkyl, alkylthio, sulfonyl, C.sub.1-C.sub.6
straight or branched chain alkyl, C.sub.2-C.sub.6 straight or
branched chain alkenyl or alkynyl, aryl, aralkyl, heteroaryl,
carbocycle, or heterocycle; provided that when R is methyl, and D
is a bond, then X is not phenyl, 4-nitrophenyl, 4-phenylazo-phenyl,
or 3,5-dinitrophenyl; when R is a substituted benzopyran group, and
D is a bond, ethenyl, or --NH--, then X is not phenyl, or
3,4,5-trihydroxyphenyl; when R is ethenyl, and D is ethenyl, then X
is not 4-hydroxy-3-methoxyphenyl; when R is methyl, and D is
ethenyl, then X is not 2-hydroxyphenyl; when R is
1-hydroxy-2-alkylamino-ethyl, and D is a bond, then X is not
phenyl, methylphenyl, or 4-methoxyphenyl; and when R is propenyl,
and D is a bond, then X is not phenyl.
2. The compound of claim 1, wherein R is a hydrophobic group.
3. The compound of claim 1, wherein R is a C.sub.1-C.sub.10
straight or branched chain alkyl.
4. The compound of claim 1, wherein X is an aryl group.
5. The compound of claim 4, wherein the aryl group is phenyl.
6. The compound of claim 4, wherein said aryl group is substituted
selected from the group consisting of halo, hydroxy, amino, nitro,
lower alkyl, dimethylamino, acetamide, sulfonyl, aryl, aralkyl,
arylthio, --COOR.sup.1, --OR.sup.1 or --NHR.sup.1, where R.sup.1 is
hydrogen, lower alkyl, and aralkyl.
7. The compound of claim 1, wherein D is a bond.
8. The compound of claim 1, wherein said compound is selected from
the group consisting of: 37
9. The compound of claim 1, wherein said compound is selected from
the group consisting of compounds 1-102.
10. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and a compound of formula (I): 38or a
pharmaceutically acceptable salt, hydrate, ester, solvate, prodrug,
metabolite, stereoisomer, or mixtures thereof, wherein A is O or S;
R is C.sub.1-C.sub.10 straight or branched chain alkyl,
C.sub.2-C.sub.10 straight or branched chain alkenyl,
C.sub.2-C.sub.10 straight or branched chain alkynyl, aryl,
heteroaryl, carbocycle, or heterocycle; D is a bond, or a
C.sub.1-C.sub.3 straight or branched chain alkyl, C.sub.2-C.sub.3
straight or branched chain alkenyl, C.sub.2-C.sub.3 straight or
branched chain alkynyl, wherein any of the carbon atoms of said
alkyl, alkenyl, or alkynyl of D are optionally replaced with
oxygen, nitrogen, or sulfur; and X is aryl, heteroaryl, carbocycle,
or heterocycle; wherein said alkyl, alkenyl, alkynyl, aryl,
heteroaryl, carbocycle, or heterocycle of R, D, or X is optionally
substituted with one or more substituents selected from hydroxy,
halo, haloalkyl, thiocarbonyl, alkoxy, alkenoxy, alkylaryloxy,
aryloxy, arylalkyloxy, cyano, nitro, amino, imino, alkylamino,
arylamino, arylazo, arylthio, aminoalkyl, sulfhydryl, thioalkyl,
alkylthio, sulfonyl, C.sub.1-C.sub.6 straight or branched chain
alkyl, C.sub.2-C.sub.6 straight or branched chain alkenyl or
alkynyl, aryl, aralkyl, heteroaryl, carbocycle, or heterocycle;
provided that when R is methyl, and D is a bond, then X is not
phenyl, 4-nitrophenyl, 4-phenylazo-phenyl, or 3,5-dinitrophenyl;
when R is a substituted benzopyran group, and D is a bond, ethenyl,
or --NH--, then X is not phenyl, or 3,4,5-trihydroxyphenyl; when R
is ethenyl, and D is ethenyl, then X is not
4-hydroxy-3-methoxyphenyl; when R is methyl, and D is ethenyl, then
X is not 2-hydroxyphenyl; when R is 1-hydroxy-2-alkylamino-ethyl,
and D is a bond, then X is not phenyl, methylphenyl, or
4-methoxyphenyl; and when R is propenyl, and D is a bond, then X is
not phenyl.
11. The pharmaceutical composition of claim 10, wherein said
compound has an IC.sub.50 of 100 .mu.M or lower for inhibiting
poly(ADP-ribose) polymerase in vitro.
12. The pharmaceutical composition of claim 10, wherein said
compound has an IC.sub.50 of 25 .mu.M or lower for inhibiting
poly(ADP-ribose) polymerase in vitro.
13. The pharmaceutical composition of claim 10, wherein the carrier
is a sterile solution, suspension or emulsion, in a single or
divided dose.
14. The pharmaceutical composition of claim 10, wherein the carrier
is a capsule or tablet containing a single or divided dose of said
compound.
15. The pharmaceutical composition of claim 10, wherein the carrier
comprises a biodegradable polymer.
16. The pharmaceutical composition of claim 15, wherein the
biodegradable polymer releases the compound of formula I over a
prolonged period of time.
17. The pharmaceutical composition of claim 10, wherein the carrier
is a solid implant.
18. The pharmaceutical composition of claim 10 for treating
diseases and disorders, wherein the diseases or disorders are
selected from the group consisting of tissue damage resulting from
cell damage or death due to necrosis or apoptosis, neuronal
mediated tissue damage or diseases, neural tissue damage resulting
from ischemia and reperfusion injury, neurological disorders and
neurodegenerative diseases, vascular stroke, cardiovascular
disorders, age-related macular degeneration, AIDS and other immune
diseases, arthritis, atherosclerosis, cachexia, cancer,
degenerative diseases of skeletal muscle involving replicative
senescence, diabetes, head trauma, immune senescence, inflammatory
bowel disorders, muscular dystrophy, osteoarthritis, osteoporosis,
chronic pain, acute pain, neuropathic pain, nervous insult,
peripheral nerve injury, renal failure, retinal ischemia, septic
shock, and skin aging, diseases or disorders relating to lifespan
or proliferative capacity of cells, and diseases or disease
conditions induced or exacerbated by cellular senescence.
19. The pharmaceutical composition of claim 18, wherein the
neurological disorder is selected from the group consisting of
peripheral neuropathy caused by physical injury or disease state,
traumatic brain injury, physical damage to the spinal cord, stroke
associated with brain damage, and demyelinating diseases.
20. The pharmaceutical composition of claim 18, wherein the
cardiovascular disorder is selected from the group consisting of
cardiovascular tissue damage, coronary artery disease, myocardial
infarction, angina pectoris and cardiogenic shock.
21. A method of inhibiting PARP activity, treating or preventing
diseases or disorders, altering gene expression, or
radiosensitizing, comprising: administering a therapeutically
effective amount of a compound of formula I: 39or a
pharmaceutically acceptable salt, hydrate, ester, solvate, prodrug,
metabolite, stereoisomer, or mixtures thereof, wherein A is O or S;
R is C.sub.1-C.sub.10 straight or branched chain alkyl,
C.sub.2-C.sub.10 straight or branched chain alkenyl,
C.sub.2-C.sub.10 straight or branched chain alkynyl, aryl,
heteroaryl, carbocycle, or heterocycle; D is a bond, or a
C.sub.1-C.sub.3 straight or branched chain alkyl, C.sub.2-C.sub.3
straight or branched chain alkenyl, C.sub.2-C.sub.3 straight or
branched chain alkynyl, wherein any of the carbon atoms of said
alkyl, alkenyl, or alkynyl of D are optionally replaced with
oxygen, nitrogen, or sulfur; and X is aryl, heteroaryl, carbocycle,
or heterocycle; wherein said alkyl, alkenyl, alkynyl, aryl,
heteroaryl, carbocycle, or heterocycle of R, D, or X is optionally
substituted with one or more substituents selected from hydroxy,
halo, haloalkyl, thiocarbonyl, alkoxy, alkenoxy, alkylaryloxy,
aryloxy, arylalkyloxy, cyano, nitro, amino, imino, alkylamino,
arylamino, arylazo, arylthio, aminoalkyl, sulfhydryl, thioalkyl,
alkylthio, sulfonyl, C.sub.1-C.sub.6 straight or branched chain
alkyl, C.sub.2-C.sub.6 straight or branched chain alkenyl or
alkynyl, aryl, aralkyl, heteroaryl, carbocycle, or heterocycle;
provided that when R is methyl, and D is a bond, then X is not
phenyl, 4-nitrophenyl, 4-phenylazo-phenyl, or 3,5-dinitrophenyl;
when R is a substituted benzopyran group, and D is a bond, ethenyl,
or --NH--, then X is not phenyl, or 3,4,5-trihydroxyphenyl; when R
is ethenyl, and D is ethenyl, then X is not
4-hydroxy-3-methoxyphenyl; when R is methyl, and D is ethenyl, then
X is not 2-hydroxyphenyl; when R is 1-hydroxy-2-alkylamino-ethyl,
and D is a bond, then X is not phenyl, methylphenyl, or
4-methoxyphenyl; and when R is propenyl, and D is a bond, then X is
not phenyl.
22. The method of claim 21, wherein the diseases or disorders are
selected from the group consisting of tissue damage resulting from
cell damage or death due to necrosis or apoptosis, neuronal
mediated tissue damage or diseases, neural tissue damage resulting
from ischemia and reperfusion injury, neurological disorders and
neurodegenerative diseases, vascular stroke, cardiovascular
disorders, age-related macular degeneration, AIDS and other immune
diseases, arthritis, atherosclerosis, cachexia, cancer,
degenerative diseases of skeletal muscle involving replicative
senescence, diabetes, head trauma, immune senescence, inflammatory
bowel disorders, muscular dystrophy, osteoarthritis, osteoporosis,
chronic pain, acute pain, neuropathic pain, nervous insult,
peripheral nerve injury, renal failure, retinal ischemia, septic
shock, and skin aging, diseases or disorders relating to lifespan
or proliferative capacity of cells, and diseases or disease
conditions induced or exacerbated by cellular senescence.
23. The method of claim 22, wherein the neurological disorder is
selected from the group consisting of peripheral neuropathy caused
by physical injury or disease state, traumatic brain injury,
physical damage to the spinal cord, stroke associated with brain
damage, and demyelinating diseases.
24. The method of claim 23, wherein the demyelinating disease is
multiple sclerosis.
25. The method of claim 22, wherein the neurodegenerative disease
is selected from the group consisting of Alzheimer's Disease,
Parkinson's Disease, Huntington's Disease and amyotropic lateral
sclerosis.
26. The method of claim 22, wherein the cancer is selected from the
group consisting of ACTH-producing tumors, acute lymphocytic
leukemia, acute nonlymphocytic leukemia, cancer of the adrenal
cortex, bladder cancer, brain cancer, breast cancer, cervix cancer,
chronic lymphocytic leukemia, chronic myelocytic leukemia,
colorectal cancer, cutaneous T-cell lymphoma, endometrial cancer,
esophageal cancer, Ewing's sarcoma, gallbladder cancer, hairy cell
leukemia, head & neck cancer, Hodgkin's lymphoma, Kaposi's
sarcoma, kidney cancer, liver cancer, lung cancer (small and/or
non-small cell), malignant peritoneal effusion, malignant pleural
effusion, melanoma, mesothelioma, multiple myeloma, neuroblastoma,
non-Hodgkin's lymphoma, osteosarcoma, ovary cancer, ovary (germ
cell) cancer, prostate cancer, pancreatic cancer, penile cancer,
retinoblastoma, skin cancer, soft-tissue sarcoma, squamous cell
carcinomas, stomach cancer, testicular cancer, thyroid cancer,
trophoblastic neoplasms, cancer of the uterus, vaginal cancer,
cancer of the vulva and Wilm's tumor.
27. The method of claim 22, wherein the bowel disorder is
colitis.
28. The method of claim 22, wherein the bowel disorder is Crohn's
disease.
29. The method of claim 22, wherein the cardiovascular disorder is
selected from the group consisting of cardiovascular tissue damage,
coronary artery disease, myocardial infarction, angina pectoris and
cardiogenic shock.
30. The method of claim 22, wherein the septic shock is endotoxic
shock.
31. The method of claim 22, wherein the disease or disease
condition induced or exacerbated by cellular senescence is selected
from the group consisting of skin aging, Alzheimer's disease,
atherosclerosis, osteoarthritis, osteoporosis, muscular dystrophy,
age-related macular degeneration, immune senescence, and AIDS.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to inhibitors of the nucleic
enzyme poly(adenosine 5'-diphospho-ribose) polymerase
["poly(ADP-ribose) polymerase" or "PARP", which is also sometimes
called "PARS" for poly(ADP-ribose) synthetase or "PART" for
poly(ADP-ribose) transferase]. More particularly, the invention
relates to the use of PARP inhibitors to prevent and/or treat
tissue damage resulting from cell damage or death due to necrosis
or apoptosis, neural tissue damage resulting from ischemia and
reperfusion injury, neurological disorders and neurodegenerative
diseases; to prevent or treat vascular stroke; to treat or prevent
cardiovascular disorders; to treat other conditions and/or
disorders such as age-related macular degeneration, AIDS and other
immune diseases, arthritis, atherosclerosis, cachexia, cancer,
degenerative diseases of skeletal muscle involving replicative
senescence, diabetes, head trauma, immune senescence, inflammatory
bowel disorders (such as colitis and Crohn's disease), muscular
dystrophy, osteoarthritis, osteoporosis, chronic and acute pain
(such as neuropathic pain), renal failure, retinal ischemia, septic
shock (such as endotoxic shock), and skin aging; to extend the
lifespan and proliferative capacity of cells; to alter gene
expression of senescent cells; or to radiosensitize hypoxic tumor
cells.
[0003] 2. Description of the Prior Art
[0004] Poly(ADP-ribose) polymerase ("PARP") is a type of enzyme
located in the nuclei of cells of various organs, including muscle,
heart and brain cells. Several structural variants or isoforms of
PARP enzymes have been isolated in various species and tissue
types, and all of these enzymes are capable of PARP activity which
consists of ADP-ribosylation. Babiychuk et al., "Higher Plants
Possess Two Structurally Different Poly(ADP-Ribose) Polymerases",
(1998) Plant Journal 15:635-645. Smith et al., "Tankyrase, a
Poly(ADP-Ribose) Polymerase at Human Telomeres", Science
282:1484-1487 (1998). These structurally-variant forms of PARP
enzymes are all referred to herein as PARP. Furthermore, the
compounds of the present invention would be expected to inhibit the
PARP activity of any and all enzymes which can perform
ADP-Ribosylation. These PARP enzymes, collectively referred to as
PARP, play a physiological role in the repair of strand breaks in
DNA. Once activated by damaged DNA fragments, PARP catalyzes the
attachment of up to 100 ADP-ribose units to a variety of nuclear
proteins, including histones and PARP itself. While the exact range
of functions of PARP has not been fully established, this enzyme is
thought to play a role in enhancing DNA repair.
[0005] During major cellular stresses, however, the extensive
activation of PARP can rapidly lead to cell damage or death through
depletion of energy stores. Four molecules of ATP are consumed for
every molecule of NAD (the source of ADP-ribose) regenerated. Thus,
NAD, the substrate of PARP, is depleted by massive PARP activation
and, in the efforts to re-synthesize NAD, ATP may also be
depleted.
[0006] It has been reported that PARP activation plays a key role
in both glutamate- and NO-induced neurotoxicity, as shown by the
use of PARP inhibitors to prevent such toxicity cortical cultures
in proportion to their potencies as inhibitors of this enzyme
(Zhang et al., "Nitric Oxide Activation of Poly(ADP-Ribose)
Synthetase in Neurotoxicity", Science, 263:687-89 (1994)); and in
hippocampal slices (Wallis et al., "Neuroprotection Against Nitric
Oxide Injury with Inhibitors of ADP-Ribosylation", NeuroReport,
5:3, 245-48 (1993)). The potential role of PARP inhibitors in
treating neurodegenerative diseases and head trauma has thus been
known. Research, however, continues to pinpoint the exact
mechanisms of their salutary effect in cerebral ischemia, (Endres
et al., "Ischemic Brain Injury is Mediated by the Activation of
Poly(ADP-Ribose) Polymerase", J. Cereb. Blood Flow Metabol., 17
7:1143-51 (1997)) and in traumatic brain injury (Wallis et al.,
"Traumatic Neuroprotection with Inhibitors of Nitric Oxide and
ADP-Ribosylation, Brain Res., 710:169-77 (1996)).
[0007] It has been demonstrated that single injections of PARP
inhibitors have reduced the infarct size caused by ischemia and
reperfusion of the heart or skeletal muscle in rabbits. In these
studies, a single injection of the PARP inhibitor,
3-amino-benzamide (10 mg/kg), either one minute before occlusion or
one minute before reperfusion, caused similar reductions in infarct
size in the heart (32-42%). Another PARP inhibitor,
1,5-dihydroxyisoquinoline (1 mg/kg), reduced infarct size by a
comparable degree (38-48%). Thiemermannn et al., "Inhibition of the
Activity of Poly(ADP Ribose) Synthetase Reduces
Ischemia-Reperfusion Injury in the Heart and Skeletal Muscle",
Proc. Natl. Acad. Sci. USA, 94:679-83 (1997). This finding has
suggested that PARP inhibitors might be able to salvage previously
ischemic heart or skeletal muscle tissue.
[0008] PARP activation has also been shown to provide an index of
damage following neurotoxic insults by glutamate (via NMDA receptor
stimulation), reactive oxygen intermediates, amyloid
.beta.-protein, n-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP)
and its active metabolite N-methyl-phenylpyridine (MPP.sup.+),
which participate in pathological conditions such as stroke,
Alzheimer's disease and Parkinson's disease. Zhang et al.,
"Poly(ADP-Ribose) Synthetase Activation: An Early Indicator of
Neurotoxic DNA Damage", J. Neurochem., 65:3, 1411-14 (1995). Other
studies have continued to explore the role of PARP activation in
cerebellar granule cells in vitro and in MPTP neurotoxicity. Cosi
et al., "Poly(ADP-Ribose) Polymerase (PARP) Revisited. A New Role
for an Old Enzyme: PARP Involvement in Neurodegeneration and PARP
Inhibitors as Possible Neuroprotective Agents", Ann. N.Y. Acad.
Sci., 825:366-79 (1997); and Cosi et al., "Poly(ADP-Ribose)
Polymerase Inhibitors Protect Against MPTP-induced Depletions of
Striatal Dopamine and Cortical Noradrenaline in C57B1/6 Mice",
Brain Res., 729:264-69 (1996).
[0009] Neural damage following stroke and other neurodegenerative
processes is thought to result from a massive release of the
excitatory neurotransmitter glutamate, which acts upon the
N-methyl-D-aspartate (NMDA) receptors and other subtype receptors.
Glutamate serves as the predominate excitatory neurotransmitter in
the central nervous system (CNS). Neurons release glutamate in
great quantities when they are deprived of oxygen, as may occur
during an ischemic brain insult such as a stroke or heart attack.
This excess release of glutamate in turn causes over-stimulation
(excitotoxicity) of N-methyl-D-aspartate (NMDA), AMPA, Kainate and
MGR receptors. When glutamate binds to these receptors, ion
channels in the receptors open, permitting flows of ions across
their cell membranes, e.g., Ca.sup.2+ and Na.sup.+ into the cells
and K.sup.+ out of the cells. These flows of ions, especially the
influx of Ca.sup.2+, cause overstimulation of the neurons. The
over-stimulated neurons secrete more glutamate, creating a feedback
loop or domino effect which ultimately results in cell damage or
death via the production of proteases, lipases and free radicals.
Excessive activation of glutamate receptors has been implicated in
various neurological diseases and conditions including epilepsy,
stroke, Alzheimer's disease, Parkinson's disease, Amyotrophic
Lateral Sclerosis (ALS), Huntington's disease, schizophrenia,
chronic pain, ischemia and neuronal loss following hypoxia,
hypoglycemia, ischemia, trauma, and nervous insult. Recent studies
have also advanced a glutamatergic basis for compulsive disorders,
particularly drug dependence. Evidence includes findings in many
animal species, as well as, in cerebral cortical cultures treated
with glutamate or NMDA, that glutamate receptor antagonists block
neural damage following vascular stroke. Dawson et al., "Protection
of the Brain from Ischemia", Cerebrovascular Disease, 319-25 (H.
Hunt Batjer ed., 1997). Attempts to prevent excitotoxicity by
blocking NMDA, AMPA, Kainate and MGR receptors have proven
difficult because each receptor has multiple sites to which
glutamate may bind. Many of the compositions that are effective in
blocking the receptors are also toxic to animals. As such, there is
no known effective treatment for glutamate abnormalities.
[0010] The stimulation of NMDA receptors, in turn, activates the
enzyme neuronal nitric oxide synthase (NNOS), which causes the
formation of nitric oxide (NO) , which more directly mediates
neurotoxiclty. Protection against glutamate neurotoxicity mediated
through the NMDA receptors has occurred following treatment with
NOS inhibitors. See Dawson et al., "Nitric Oxide Mediates Glutamate
Neurotoxicity in Primary Cortical Cultures", Proc. Nati. Acad. Sci.
USA, 88:6368-71 (1991); and Dawson et al., "Mechanisms of Nitric
Oxide-mediated Neurtoxicity in Primary Brain Cultures", J.
Neurosci., 13:6, 2651-61 (1993). Protection against glutamate
neurotoxicity mediated through NMDA receptors can also occur in
cortical cultures from mice with targeted disruption of NNOS. See
Dawson et al., "Resistance to Neurotoxicity in Cortical Cultures
from Neuronal Nitric Oxide Synthase-Deficient Mice", J. Neurosci.,
16:8, 2479-87 (1996),
[0011] It is known that neural damage following vascular stroke is
markedly diminished in animals treated with NOS inhibitors or in
mice with NNOS gene disruption. Iadecola, "Bright and Dark Sides of
Nitric Oxide in Ischemic Brain Injury", Trends Neurosci., 20:3,
132-39 (1997); and Huang et al., "Effects of Cerebral Ischemia in
Mice Deficient in Neuronal Nitric Oxide Synthase", Science,
265:1883-85 (1994) See also, Beckman et al., "Pathological
Implications of Nitric Oxide, Superoxide and Peroxynitrite
Formation", Biochem. Soc. Trans., 21:330-34 (1993). Either NO or
peroxynitrite can cause DNA damage, which activates PARP. Further
support for this is provided in Szab et al., "DNA Strand Breakage,
Activation of Poly(ADP-Ribose) Synthetase, and Cellular Energy
Depletion are Involved in the Cytotoxicity in Macrophages and
Smooth Muscle Cells Exposed to Peroxynitrite", Proc. Natl Acad.
Sci. USA, 93:1753-58 (1996).
[0012] Zhang et al., U.S. Pat. No. 5,587,384 issued Dec. 24, 1996,
discusses the use of certain PARP inhibitors, such as benzamide and
1,5-dihydroxy-isoquinoline, to prevent NMDA-receptor mediated
neurotoxicity and, thus, treat stroke, Alzheimer's disease,
Parkinson's disease and Huntington's disease. However, it is has
now been discovered that Zhang et al. may have been in error in
classifying neurotoxicity as NMDA-receptor mediated neurotoxicity.
Rather, the in vivo neurotoxicity present is more appropriately
classified as glutamate neurotoxicity. See Zhang et al. "Nitric
Oxide Activation of Poly(ADP-Ribose) Synthetase in Neurotoxicity",
Science, 263:687-89 (1994). See also, Cosi et al., Poly(ADP-Ribose)
Polymerase Inhibitors Protect Against MPTP-induced Depletions of
Striatal Dopamine and Cortical Noradrenaline in C57B1/6 Mice",
Brain Res., 729:264-69 (1996).
[0013] It is also known that PARP inhibitors affect DNA repair
generally. Cristovao et al., "Effect of a Poly(ADP-Ribose)
Polymerase Inhibitor on DNA Breakage and Cytotoxicity Induced by
Hydrogen Peroxide and .gamma.-Radiation," Terato., Carcino., and
Muta., 16:219-27 (1996), discusses the effect of hydrogen peroxide
and .gamma.-radiation on DNA strand breaks in the presence of and
in the absence of 3-aminobenzamide, a potent inhibitor of PARP.
Cristovao et al. observed a PARP-dependent recovery of DNA strand
breaks in leukocytes treated with hydrogen peroxide.
[0014] PARP inhibitors have been reported to be effective in
radiosensitizing hypoxic tumor cells and effective in preventing
tumor cells from recovering from potentially lethal damage of DNA
after radiation therapy, presumably by their ability to prevent DNA
repair. See U.S. Pat. Nos. 5,032,617; 5,215,738; and 5,041,653.
[0015] Evidence also exists that PARP inhibitors are useful for
treating inflammatory bowel disorders. Salzman et al., "Role of
Peroxynitrite and Poly(ADP-Ribose) Synthase Activation Experimental
Colitis," Japanese J. Pharm., 75, Supp. I:15 (1997), discusses the
ability of PARP inhibitors to prevent or treat colitis. Colitis was
induced in rats by intraluminal administration of the hapten
trinitrobenzene sulfonic acid in 50% ethanol and treated with
3-aminobenzamide, a specific inhibitor of PARP activity. Inhibition
of PARP activity reduced the inflammazory response and restored the
morphology and the energetic status of the distal colon. See also,
Southan et al., "Spontaneous Rearrangement of Amino-alkylithioureas
into Mercaptoalkylguanidines, a Novel Class of Nitric Oxide
Synthase Inhibitors with Selectivity Towards the Inducible
Isoform", Br. J. Pharm. 117:619-32 (1996); and Szab et al.,
"Mercaptoethylguanidine and Guanidine Inhibitors of Nitric Oxide
Synthase React with Peroxynitrite and Protect Against
Peroxynitrite-induced Oxidative Damage", J. Biol. Chem.,
272:9030-36 (1997).
[0016] Evidence also exists that PARP inhibitors are useful for
treating arthritis. Szab et al., "Protective Effects of an
Inhibitor of Poly(ADP-Ribose) Synthetase in Collagen-Induced
Arthritis," Japanese J. Pharm., 75, Supp. I:102 (1997), discusses
the ability of PARP inhibitors to prevent or treat collagen-induced
arthritis. See also Szab et al., "DNA Strand Breakage, Activation
of Poly(ADP-Ribose) Synthetase, and Cellular Energy Depletion are
Involved in the Cyrotoxicity in Macrophages and Smooth Muscle Cells
Exposed to Peroxynitrite," Proc. Natl. Acad. Sci. USA, 93:1753-58
(March 1996); Bauer et al., "Modification of Growth Related
Enzymatic Pathways and Apparent Loss of Tumorigenicity of a
ras-transformed Bovine Endothelial Cell Line by Treatment with
5-Iodo-6-amino-1,2-benzopyrone (INH.sub.2BP)", Intl. J. Oncol.,
8:239-52 (1996); and Hughes et al., "Induction of T Helper Cell
Hyporesponsiveness in an Experimental Model of Autoimmunity by
Using Nonmitogenic Anti-CD3 Monoclonal Antibody", J. Immuno.,
153:3319-25 (1994).
[0017] Further, PARP inhibitors appear to be useful for treating
diabetes. Heller et al., "Inactivation of the Poly(ADP-Ribose)
Polymerase Gene Affects Oxygen Radical and Nitric Oxide Toxicity in
Islet Cells," J. Biol. Chem., 270:19,-11176-80 (May 1995),
discusses the tendency of PARP to deplete cellular NAD+ and induce
the death of insulin-producing islet cells. Heller et al. used
cells from mice with inactivated PARP genes and found that these
mutant cells did not show NAD+ depletion after exposure to
DNA-damaging radicals. The mutant cells were also found to be more
resistant to the toxicity of NO.
[0018] Further still, PARP inhibitors have been shown to be useful
for treating endotoxic shock or septic shock. Zingarelli et al.,
"Protective Effects of Nicotinamide Against Nitric Oxide-Mediated
Delayed Vascular Failure in Endotoxic Shock: Potential Involvement
of PolyADP Ribosyl Synthetase," Shock, 5:258-64 (1996), suggests
that inhibition of the DNA repair cycle triggered by poly(ADP
ribose) synthetase has protective effects against vascular failure
in endotoxic shock. Zlngarelli et al. found that nicotinamide
protects against delayed, NO-mediated vascular failure in endotoxic
shock. Zingarelli et al. also found that the actions of
nicotinamide may be related to inhibition of the NO-mediated
activation of the energy-consuming DNA repair cycle, triggered by
poly(ADP ribose) synthezase. See also, Cuzzocrea, "Role of
Peroxynitrite and Activation of Poly(ADP-Ribose) Synthetase in the
Vascular Failure Induced by Zymosan-activated Plasma," Brit. J.
Pharm., 122:493-503 (1997).
[0019] Yet another known use for PARP inhibitors is treating
cancer. Suto et al., "Dihydroisoquinolinones: The Design and
Synthesis of a New Series of Potent Inhibitors of Poly(ADP-Ribose)
Polymerase", Anticancer Drug Des., 7:107-17 (1991), discloses
processes for synthesizing a number of different PARP inhibitors.
In addition, Suto et al., U.S. Pat. No. 5,177,075, discusses
several isoquinolines used for enhancing the lethal effects of
ionizing radiation or chemotherapeutic agents on tumor cells.
Weltin et al., "Effect of 6(5H)-Phenanthridinone, an Inhibitor of
Poly(ADP-ribose) Polymerase, on Cultured Tumor Cells", Oncol. Res.,
6:9, 399-403 (1994), discusses the inhibition of PARP activity,
reduced proliferation of tumor cells, and a marked synergistic
effect when tumor cells are co-treated with an alkylating drug.
[0020] Still another use for PARP inhibitors is the treatment of
peripheral nerve injuries, and the resultant pathological pain
syndrome known as neuropathic pain, such as that induced by chronic
constriction injury (CCI) of the common sciatic nerve and in which
transsynaptic alteration of spinal cord dorsal horn characterized
by hyperchromatosis of cytoplasm and nucleoplasm (so-called "dark"
neurons) occurs. See Mao et al., Pain, 72:355-366 (1997).
[0021] PARP inhibitors have also been used to extend the lifespan
and proliferative capacity of cells including treatment of diseases
such as skin aging, Alzheimer's disease, atherosclerosis,
osteoarthritis, osteoporosis, muscular dystrophy, degenerative
diseases of skeletal muscle involving replicative senescence,
age-related macular degeneration, immune senescence, AIDS, and
other immune diseases; and to alter gene expression of senescent
cells. See WO 98/27975.
[0022] Large numbers of known PARP inhibitors have been described
in Banasik et al., "Specific Inhibitors of Poly(ADP-Ribose)
Synthetase and Mono(ADP-Ribosyl)-Transferase", J. Biol. Chem.,
267:3, 1569-75 (1992), and in Banasik et al., "Inhibitors and
Activators of ADP-Ribosylation Reactions", Molec. Cell. Biochem.,
138:185-97 (1994).
[0023] However, the approach of using these PARP inhibitors in the
ways discussed above has been limited in effect. For example, side
effects have been observed with some of the best-known PARP
inhibitors, as discussed in Milam et al., "Inhibitors of
Poly(Adenosine Diphosphate-Ribose) Synthesis: Effect on Other
Metabolic Processes", Science, 223:589-91 (1984). Specifically, the
PARP inhibitors 3-aminobenzamide and benzamide not only inhibited
the action of PARP but also were shown to affect cell viability,
glucose metabolism, and DNA synthesis. Thus, it was concluded that
the usefulness of these PARP inhibitors may be severely restricted
by the difficulty of finding a dose that will inhibit the enzyme
without producing additional metabolic effects.
[0024] The inventors have now discovered that select ortho-diphenol
compounds can inhibit PARP activity and can treat or prevent tissue
damage resulting from cell damage or death due to necrosis or
apoptosis and/or can ameliorate neural tissue damage, including
that following focal ischemia and reperfusion injury. Generally,
inhibition of PARP activity spares the cell from energy loss,
preventing irreversible depolarization of the neurons and, thus,
provides neuroprotection. While not wishing to be bound thereby, it
is thought that PARP activation may play a common role in still
other excitotoxic mechanisms, perhaps as yet undiscovered, in
addition to the production of free radicals and NO.
[0025] Certain related compounds have been disclosed for medical
treatments and other uses. However, these compounds are
structurally distinguishable and directed to uses which emphasize
their toxic characterstics. Compounds that are used primarily as
synthetic intermediates in the preparation of compounds for:
treating hyperlipemia in U.S. Pat. No. 5,719,303 to Yoshida et al.
"Phosphinic Acid Derivatives"; treating plant nepovirus in
"Inhibition of Tomato Ringspot Virus by Flavenoids", by Malhotra et
al, (1996) Phytochem. 43:1271-1276; inhibiting fibrosis in Japanese
Patent 92JP-0098635 to Kyota et al. "Fibrosis-inhibiting Agents
Containing Gallate Esters"; treatment of hypertension in European
Patent 86EP-194046 to Hargreaves et al "Dihydropyridine
Alkanolamines and Their Use"; bronchodilation in U.S. Pat. No.
4,336,400 to Minatoya et a.. "3-(Hydroxy or
Hydroxymethyl)-4-Hydroxy-alpha (Aminomethyl) Benzyl Alcohols and
Methods of Use"; treating snakebite in U.S. Pat. No. 4,124,724 to
Agoro, "Crystalline Caffeic Acid Derivatives and Compositions for
Treating Snakebite"; and bronchodilation in "Esters of
N-tert-butylarterenol: Long-acting New Bronchodilators with Reduced
Cardiac Effects", by Tullar et al, (1976) J. Med. Chem.
19:834-838.
[0026] Other related, but structurally distinct compounds for
unrelated uses include: PCT Publication WO9720944 by Banister et
al., "Melanin Production"; "(+) -Catechin: Benzoyl Protection of OH
Groups and NMR Study of Products", by Marek et al, (1997) Chem.
Pap. 51:107-110; "Decomposition of Polyurethane Foams Derived from
Condensed Tannin", by Ge et al, (1996) Mokuzai Gakkaishi
42:776-781; "Cinnamic Acid Derivatives", Yamaguchi et al, (1996)
Sekiyu Gakkaishi 39:273-278; Japanese Patent JP03088882 to Kanba et
al., "Weather Resistant Water-Based Fluoropolymer Coating
Compositions"; "A DTA Study of Phenols: III Polyhydroxyphenols and
Naphthols", by Buckman et al, (1991) J. Therm. Anal. 37:79-94; and
U.S. Pat. No. 3,904,671 to Minatoya et al. "(Aminohydroalkyl)
Catechol Diesters".
[0027] Accordingly, there remains a need for compounds capable of
acting as PARP inhibitors, pharmaceutical compositions containing
the same and methods of using the same that produce more potent and
reliable effects, particularly with respect to treatment of tissue
damage resulting from cell death or damage due to necrosis or
apoptosis, and less side effects.
SUMMARY OF THE INVENTION
[0028] The present invention relates to novel poly(ADP-ribose)
polymerase ("PARP") inhibitors and methods for using the same
including effecting a neurona activity in an animal. As such, they
may treat or prevent neural tissue damage resulting from cell
damage or death due to necrosis or apoptosis, cerebral ischemia and
reperfusion injury or neurodegenerative diseases in an animal; they
may extend the lifespan and proliferative capacity of cells and
thus be used to treat or prevent diseases associated therewith;
they may alter gene expression of senescent cells; and they may
radiosensitize hypoxic tumor cells. Preferably, the compounds of
the invention treat or revent tissue damage resulting from cell
damage or death due to necrosis or apoptosis, and/or effect
neuronal activity, either mediated or not mediated by glutamate
neurotoxicity. These compounds are thought to interfere with more
than the glutamate neurotoxicity and NO-mediated biological
pathways. Further, the compounds of the invention can treat or
prevent other tissue damage related to PARP activation.
[0029] For example, the compounds of the invention can treat or
prevent cardiovascular tissue damage resulting from cardiac
ischemia or reperfusion injury. Reperfusion injury, for instance,
occurs at the termination of cardiac bypass procedures or during
cardiac arrest when the heart, once prevented from receiving blood,
begins to reperfuse.
[0030] The compounds of the present invention can also be used to
extend or increase the lifespan or proliferation of cells and thus
to treat or prevent diseases associated therewith and induced or
exacerbated by cellular senescence including skin aging,
atherosclerosis, osteoarthritis, osteoporosis, muscular dystrophy,
degenerative diseases of skeletal muscle involving replicative
senescence, age-related macular degeneration, immune senescence,
AIDS and other immune diseases, and other diseases associated with
cellular senescence and aging, as well as to alter the gene
expression of senescent cells. These compounds can also be used to
treat cancer and to radiosensitize hypoxic tumor cells to render
the tumor cells more susceptible to radiation therapy and to
prevent the tumor cells from recovering from potentially lethal
damage of DNA after radiation therapy, presumably by their ability
to prevent DNA repair. The compounds of the present invention can
be used to prevent or treat vascular stroke; to treat or prevent
cardiovascular disorders; to treat other conditions and/or
disorders such as age-related macular degeneration, AIDS and other
immune diseases, arthritis, atherosclerosis, cachexia, cancer,
degenerative diseases of skeletal muscle involving replicative
senescence, diabetes, head trauma, immune senescence, inflammatory
bowel disorders (such as colitis and Crohn's disease), muscular
dystrophy, osteoarthritis, osteoporosis, chronic and/or acute pain
(such as neuropathic pain), renal failure, retinal ischemia, septic
shock (such as endotoxic shock), and skin aging. Preferably, the
compounds of the invention exhibit an IC.sub.50 for inhibiting PARP
in vitro of about 100 .mu.M or lower, more preferably, about 25
.mu.M or lower, more preferably, about 10 .mu.M or lower, and most
preferably, about 1 .mu.M or lower.
[0031] Specifically, the present invention relates to a compound of
formula I: 2
[0032] or a pharmaceutically acceptable salt, hydrate, ester,
solvate, prodrug, metabolite, stereoisomer, or mixtures thereof,
wherein
[0033] A is O or S;
[0034] R is C.sub.1-C.sub.10 straight or branched chain alkyl,
C.sub.2-C.sub.10 straight or branched chain alkenyl,
C.sub.2-C.sub.10 straight or branched chain alkynyl, aryl,
heteroaryl, carbocycle, or heterocycle;
[0035] D is a bond, or a C.sub.1-C.sub.3 straight or branched chain
alkyl, C.sub.2-C.sub.3 straight or branched chain alkenyl,
C.sub.2-C.sub.3 straight or branched chain alkynyl, wherein any of
the carbon atoms of said alkyl, alkenyl, or alkynyl of D are
optionally replaced with oxygen, nitrogen, or sulfur; and
[0036] X is aryl, heteroaryl, carbocycle, or heterocycle;
[0037] wherein said alkyl, alkenyl, alkynyl, aryl, heteroaryl,
carbocycle, or heterocycle of R, D, or X is optionally substituted
with one or more substituents selected from hydroxy, halo,
haloalkyl, thiocarbonyl, alkoxy, alkenoxy, alkylaryloxy, aryloxy,
arylalkyloxy, cyano, nitro, amino, imino, alkylamino, arylamino,
arylazo, arylthio, aminoalkyl, sulfhydryl, thioalkyl, alkylthio,
sulfonyl, C.sub.1-C.sub.6 straight or branched chain alkyl,
C.sub.2-C.sub.6 straight or branched chain alkenyl or alkynyl,
aryl, aralkyl, heteroaryl, carbocycle, or heterocycle;
[0038] provided that when R is methyl, and D is a bond, then X is
not phenyl, 4-nitrophenyl, 4-phenylazo-phenyl, or
3,5-dinitrophenyl; when R is a substituted benzopyran group, and D
is a bond, ethenyl, or --NH--, then X is not phenyl, or
3,4,5-trihydroxyphenyl; when R is ethenyl, and D is ethenyl, then X
is not 4-hydroxy-3-methoxyphenyl; when R is methyl, and D is
ethenyl, then X is not 2-hydroxyphenyl; when R is
1-hydroxy-2-alkylamino-ethyl, and D is a bond, then X is not
phenyl, methylphenyl, or 4-methoxyphenyl; and when R is propenyl,
and D is a bond, then X is not phenyl.
[0039] A preferred embodiment of this invention is the compound of
formula I, wherein R is a hydrophobic group. Another preferred
embodiment of the invention is the compound of formula I, wherein R
is a C.sub.1-C.sub.10 straight or branched chain alkyl.
[0040] The following are particularly preferred compounds of the
present invention: 3
[0041] Additional preferred compounds are those of formula I as
follows where A is O as exemplified by Compounds 1-51: 4
[0042] as in Table I as follows:
1TABLE I where A is O Compound R D X 1 methyl bond 4-bromophenyl 2
ethyl bond phenyl 3 n-propyl bond 3,4,5-trihydroxy- phenyl 4
i-propyl bond 3,4,5-trimethoxy- phenyl 5 n-butyl bond
3-hydroxyphenyl 6 t-butyl bond 4-nitro-naphthyl 7 s-butyl bond
3-hydroxy- naphthyl 8 pentyl bond benzyl 9 hexyl bond 4-ethylphenyl
10 heptyl bond 4-ethenylphenyl 11 octyl bond 4-quinolyl 12 nonyl
bond 2-thiazolyl 13 decyl bond 3-furyl 14 1,1,dimethylpropyl bond
phenyl 15 ethenyl bond cyclohexyl 16 prop-2-enyl bond
3-bromocyclohexyl 17 phenyl bond adamantyl 18 naphthyl bond
4-indolyl 19 4-nitrophenyl bond 2-imidazolyl 20 4-hydroxyphenyl
bond 1-naphthyl 21 4-chlorophenyl bond 4-nitrophenyl 22
4-methylphenyl bond 4-hydroxyphenyl 23 4-methoxyphenyl bond
3-piperidyl 24 4-dimethylamino- bond 3,4,5-trimethyl- phenyl phenyl
25 phenyl-ethyl-phenyl bond 3-pyridyl 26 4-nitro-3-hydroxy- bond
3,4,5-trifluoro- phenyl phenyl 27 1-pyridyl bond 1-pyrrolidyl 28
1-piperidyl bond 4-phenylazo- phenyl 29 1-pyrrolidyl 2-bromo-
4-amino-3- propyl hydroxy-phenyl 30 cyclohexyl prop-2-
3,4,5-triamino- enyl phenyl 31 cyclopentyl methyl 4-hydroxyphenyl
32 adamantyl ethyl phenyl 33 benzyl i-propyl 9-anthracenyl 34
4-hydroxybenzyl n-propyl 4-pyrenyl 35 3,4,5-trihydroxy- 2-imino-
3-furyl phenyl propyl 36 thiazolyl 2-thio- 3-thiophenyl propyl 37
2-phenylethyl 3-sulfonyl- 4-pyrimidinyl propyl 38 3-phenylpropyl
ethenyl 4-isoquinolyl 39 2-phenylethenyl bond 4-sulfonylphenyl 40
3-phenylprop-2-enyl chloro- 4-imino-phenyl methyl 41 3-bromophenyl
--CH.sub.2--N.dbd.CH-- 4-phenylethoxy- phenyl 42 4-fluoro-n-butyl
--CH.sub.2--S--CH.sub.2-- 4-ethylphenoxy- phenyl 43 3-methoxypropyl
--CH.sub.2--NH--CH.sub.2-- 4-phenoxy-phenyl 44 2-hydroxyethyl
--CH.sub.2--O--CH.sub.2-- 3-phenylpropyl- phenyl 45 tert-butyl
--CH.sub.2-- 5 46 tert-butyl bond 2-chloro-phenyl 47 tert-butyl
bond 4-chloro-phenyl 48 tert-butyl bond 3,4,5-trimethoxy- phenyl 49
tert-butyl bond 6 50 tert-butyl bond 7 51 tert-butyl
--O--CH.sub.2--, phenyl X attaches directly to the CH.sub.2
[0043] Additional preferred compounds are those of formula I as
follows where A is S as exemplified by Compounds 52-102: 8
[0044] as shown in Table II as follows:
2TABLE II where A is S Compound R D X 52 methyl bond 4-bromophenyl
53 ethyl bond phenyl 54 n-propyl bond 3,4,5-trihydroxy- phenyl 55
i-propyl bond 3,4,5-trimethoxy- phenyl 56 n-butyl bond
3-hydroxyphenyl 57 t-butyl bond 4-nitro-naphthyl 58 s-butyl bond
3-hydroxy- naphthyl 59 pentyl bond benzyl 60 hexyl bond
4-ethylphenyl 61 heptyl bond 4-ethenylphenyl 62 octyl bond
4-quinolyl 63 nonyl bond 2-thiazolyl 64 decyl bond 3-furyl 65
1,1,dimethylpropyl bond phenyl 66 ethenyl bond cyclohexyl 67
prop-2-enyl bond 3-bromocyclohexyl 68 phenyl bond adamantyl 69
naphthyl bond 4-indolyl 70 4-nitrophenyl bond 2-imidazolyl 71
4-hydroxyphenyl bond 1-naphthyl 72 4-chlorophenyl bond
4-nitrophenyl 73 4-methylphenyl bond 4-hydroxphenly 74
4-methoxyphenyl bond 3-piperidyl 75 4-dimethylamino- bond
3,4,5-trimethyl- phenyl phenyl 76 phenyl-ethyl-phenyl bond
3-pyridyl 77 4-nitro-3-hydroxy- bond 3,4,5-trifluoro- phenyl phenyl
78 1-pyridyl bond 1-pyrroldyl 79 1-piperidyl bond 4-phenylazo-
phenyl 80 1-pyrrolidyl 2-bromo- 4-amino-3- propyl hydroxy-phenyl 81
cyclohexyl prop-2- 3,4,5-triamino- enyl phenyl 82 cyclopentyl
methyl 4-hydroxyphenyl 83 adamantyl ethyl phenyl 84 benzyl i-propyl
9-anthracenyl 85 4-hydroxybenzyl n-propyl 4-pyrenyl 86
3,4,5-trihydroxy- 2-imino- 3-furyl phenyl propyl 87 thiazolyl
2-thio- 3-thiophenyl propyl 88 2-phenylethyl 2-sulfonyl-
4-pyrimidinyl propyl 89 3-phenylpropyl ethenyl 4-isoquinolyl 90
2-phenylethenyl bond 4-sulfonylphenyl 91 3-phenylprop-2-enyl
chloro- 4-imino-phenyl methyl 92 3-bromophenyl
--CH.sub.2--N.dbd.CH-- 4-phenylethoxy- phenyl 93 4-fluoro-n-butyl
--CH.sub.2--S--CH.sub.2-- 4-ethylphenoxy- phenyl 94 3-methoxypropyl
--CH.sub.2NH--CH.sub.2-- 4-phenoxy-phenyl 95 2-hydroxyethyl
--CH.sub.2--O--CH.sub.2-- 3-phenylpropyl- phenyl 96 tert-butyl
--CH.sub.2-- 9 97 tert-butyl bond 2-chloro-phenyl 98 tert-butyl
bond 4-chloro-phenyl 99 tert-butyl bond 3,4,5-trimethoxy- phenyl
100 tert-butyl bond 10 101 tert-butyl bond 11 102 tert-butyl
--O--CH.sub.2--, phenyl X attaches directly to the CH.sub.2
BRIEF DESCRIPTION OF THE DRAWINGS
[0045] FIG. 1 shows the distribution of the cross-sectional infarct
area at representative levels along the rostrocaudal axis, as
measured from the interaural line in non-treated animals and in
animals treated with 10 mg/kg of
3,4-dihydro-5-[4-(1piperidinyl)-botoxyl]-1(2H)-isoquinolinone.
[0046] FIG. 2 shows the effect of intraperitoneal administration of
3,4-dihydro-5-[4-(1-piperidinyl)-butoxy]-1(2H)-isoquinolinone on
the infarct volume.
DETAILED DESCRIPTION OF THE INVENTION
[0047] The present invention pertains to compounds, pharmaceutical
compositions containing the same, methods of using the same, and
process of making the same, wherein such compounds are useful as
inhibitors of poly(ADP-ribose) polymerase (PARP). As such, they may
treat or prevent neural tissue damage resulting from cell damage or
death due to necrosis or apoptosis, cerebral ischemia and
reperfusion injury or neurodegenerative diseases in an animal; they
may extend the lifespan and proliferative capacity of cells and
thus be used to treat or prevent diseases associated therewith;
they may alter gene expression of senescent cells; and they may
radiosensitize hypoxic tumor cells. Preferably, the compounds of
the invention treat or prevent tissue damage resulting from cell
damage or death due to necrosis or apoptosis, and/or effect
neuronal activity, either mediated or not mediated by glutamate
neurotoxicity. These compounds are thought to interfere with more
than the glutamate neurotoxicity and NO-mediated biological
pathways. Further, the compounds of the invention can treat or
prevent other tissue damage related to PARP activation.
[0048] For example, the compounds of the invention can treat or
prevent cardiovascular tissue damage resulting from cardiac
ischemia or reperfusion injury. Reperfusion injury, for instance,
occurs at the termination of cardiac bypass procedures or during
cardiac arrest when the heart, once prevented from receiving blood,
begins to reperfuse.
[0049] The compounds of the present invention can also be used to
extend or increase the lifespan or proliferation of cells and thus
to treat or prevent diseases associated therewith and induced or
exacerbated by cellular senescence including skin aging,
atherosclerosis, osteoarthritis, osteoporosis, muscular dystrophy,
degenerative diseases of skeletal muscle involving replicative
senescence, age-related macular degeneration, immune senescence,
AIDS and other immune diseases, and other diseases associated with
cellular senescence and aging, as well as to alter the gene
expression of senescent cells. These compounds can also be used to
treat cancer and to radiosensitize hypoxic tumor cells to render
the tumor cells more susceptible to radiation therapy and to
prevent the tumor cells from recovering from potentially lethal
damage of DNA after radiation therapy, presumably by their ability
to prevent DNA repair. The compounds of the present invention can
be used to prevent or treat vascular stroke; to treat or prevent
cardiovascular disorders; to treat other conditions and/or
disorders such as age-related macular degeneration, AIDS and other
immune diseases, arthritis, atherosclerosis, cachexia, cancer,
degenerative diseases of skeletal muscle involving replicative
senescence, diabetes, head trauma, immune senescence, inflammatory
bowel disorders (such as colitis and Crohn's disease), muscular
dystrophy, osteoarthritis, osteoporosis, chronic and/or acute pain
(such as neuropathic pain), renal failure, retinal ischemia, septic
shock (such as endotoxic shock), and skin aging.
[0050] Preferably, the compounds of the invention act as PARP
inhibitors to treat or prevent tissue damage resulting from cell
death or damage due to necrosis or apoptosis; to treat or prevent
neural tissue damage resulting from cerebral ischemia and
reperfusion injury or neurodegenerative diseases in an animal; to
extend and increase the lifespan and proliferative capacity of
cells; to alter gene expression of senescent cells; and to
radiosensitize tumor cells.
[0051] What the inventors have now discovered is that the
ortho-diphenol compounds of the present invention can act to
inhibit PART and can ameliorate neural tissue damage and
cardiovascular tissue damage, including that following focal
ischemia, myocardial infarction, and reperfusion injury. Generally,
inhibition of PARP activity spares the cell from energy loss,
preventing irreversible depolarization of the neurons and, thus,
provides neuroprotection. While not wishing to be bound thereby, it
is thought that PARP activation may play a common role in still
other excitotoxic mechanisms, perhaps as yet undiscovered, in
addition to the production of free radicals and NO. Preferably, the
compounds of the invention exhibit an IC.sub.50 for inhibiting PARP
in vitro of about 100 .mu.M or lower, more preferably, about 25
.mu.M or lower, most preferably, about 1 .mu.M or lower.
[0052] Preferred PARP inhibitors of the present invention include
compounds having formula I: 12
[0053] or a pharmaceutically acceptable salt, hydrate, ester,
solvate, prodrug, metabolite, stereoisomer, or mixtures thereof,
wherein
[0054] A is O or S;
[0055] R is C.sub.1-C.sub.10 straight or branched chain alkyl,
C.sub.2-C.sub.10 straight or branched chain alkenyl,
C.sub.2-C.sub.10 straight or branched chain alkynyl, aryl,
heteroaryl, carbocycle, or heterocycle;
[0056] D is a bond, or a C.sub.1-C.sub.3 straight or branched chain
alkyl, C.sub.2-C.sub.3 straight or branched chain alkenyl,
C.sub.2-C.sub.3 straight or branched chain alkynyl, wherein any of
the carbon atoms of said alkyl, alkenyl, or alkynyl of D are
optionally replaced with oxygen, nitrogen, or sulfur; and
[0057] X is aryl, heteroaryl, carbocycle, or heterocycle;
[0058] wherein said alkyl, alkenyl, alkynyl, aryl, heteroaryl,
carbocycle, or heterocycle of R, D, or X is optionally substituted
with one or more substituents selected from hydroxy, halo,
haloalkyl, thiocarbonyl, alkoxy, alkenoxy, alkylaryloxy, aryloxy,
arylalkyloxy, cyano, nitro, amino, imino, alkylamino, arylamino,
arylazo, arylthio, aminoalkyl, sulfhydryl, thioalkyl, alkylthio,
sulfonyl, C.sub.1-C.sub.6 straight or branched chain alkyl,
C.sub.2-C.sub.6 straight or branched chain alkenyl or alkynyl,
aryl, aralkyl, heteroaryl, carbocycle, or heterocycle;
[0059] provided that when R is methyl, and D is a bond, then X is
not phenyl, 4-nitrophenyl, 4-phenylazo-phenyl, or
3,5-dinitrophenyl; when R is a substituted benzopyran group, and D
is a bond, ethenyl, or --NH--, then X is not phenyl, or
3,4,5-trihydroxyphenyl; when R is ethenyl, and D is ethenyl, then X
is not 4-hydroxy-3-methoxyphenyl; when R is methyl, and D is
ethenyl, then X is not 2-hydroxyphenyl; when R is
1-hydroxy-2-alkylamino-ethyl, and D is a bond, then X is not
phenyl, methylphenyl, or 4-methoxyphenyl; and when R is propenyl,
and D is a bond, then X is not phenyl.
[0060] Preferred compounds of the present invention include
compounds of formula I, where R is a hydrophobic group or a
C.sub.1-C.sub.10 straight or branched chain alkyl.
[0061] Other preferred compounds of the present invention include
compounds of formula I, where X is an aryl group; particularly
where the aryl group is phenyl. The aryl group may be substituted
with at least one non-hydrogen, non-interfering substituent;
preferably a halo, hydroxy, amino, nitro, lower alkyl,
dimethylamino, acetamide, sulfonyl, aryl, aralkyl, arylthio,
--COOR.sup.1, --OR.sup.1 or --NHR.sup.1, where R.sup.1 is hydrogen,
lower alkyl, or aralkyl substituent.
[0062] Still other preferred compounds of the present invention
include compounds of formula I where D is a bond.
[0063] The following are particularly preferred compounds of the
present invention: 13
[0064] Other preferred compounds of the present invention include
compounds 1-51 listed in Table I as follows:
3TABLE I 14 Compound R D X 1 methyl bond 4-bromophenyl 2 ethyl bond
phenyl 3 n-propyl bond 3,4,5-trihydroxy- phenyl 4 i-propyl bond
3,4,5-trimethoxy- phenyl 5 n-butyl bond 3-hydroxyphenyl 6 t-butyl
bond 4-nitro-naphthyl 7 s-butyl bond 3-hydroxy- naphthyl 8 pentyl
bond benzyl 9 hexyl bond 4-ethylphenyl 10 heptyl bond
4-ethenylphenyl 11 octyl bond 4-quinolyl 12 nonyl bond 2-thiazolyl
13 decyl bond 3-furyl 14 1,1,dimethylpropyl bond phenyl 15 ethenyl
bond cyclohexyl 16 prop-2-enyl bond 3-bromocyclohexyl 17 phenyl
bond adamantyl 18 naphthyl bond 4-indolyl 19 4-nitrophenyl bond
2-imidazolyl 20 4-hydroxyphenyl bond 1-naphthyl 21 4-chlorophenyl
bond 4-nitrophenyl 22 4-methylphenyl bond 4-hydroxyphenyl 23
4-methoxyphenyl bond 3-piperidyl 24 4-dimethylamino- bond
3,4,5-trimethyl- phenyl phenyl 25 phenyl-ethyl- bond 3-pyridyl
phenyl 26 4-nitro-3-hydroxy- bond 3,4,5-trifluoro- phenyl phenyl 27
1-pyridyl bond 1-pyrrolidyl 28 1-piperidyl bond 4-phenylazo- phenyl
29 1-pyrroldyl 2-bromo- 4-amino-3- propyl hydroxy-phenyl 30
cyclohexyl prop-2- 3,4,5-triamino- enyl phenyl 31 cyclopentyl
methyl 4-hydroxyphenyl 32 adamantyl ethyl phenyl 33 benzyl i-propyl
9-anthracene 34 4-hydroxybenzyl n-propyl 4-pyrenyl 35
3,4,5-trihydroxy- 2-imino- 3-furyl phenyl propyl 36 thiazolyl
2-thio- 3-thiophenyl propyl 37 2-phenylethyl 2-sulfonyl-
4-pyrimidinyl propyl 38 3-phenylpropyl ethenyl 4-isoquinolyl 39
2-phenylethenyl bond 4-sulfonylphenyl 40 3-phenylprop-2- chloro-
4-imino-phenyl enyl methyl 41 3-bromopropyl --CH.sub.2--N.dbd.CH--
4-phenylethoxy- phenyl 42 4-fluoro-n-butyl
--CH.sub.2--S--CH.sub.2-- 4-ethylphenoxy- phenyl 43 3-methoxypropyl
--CH.sub.2--NH--CH.sub.2-- 4-phenoxy-phenyl 44 2-hydroxyethyl
--CH.sub.2--O--CH.sub.2-- 3-phenylpropyl- phenyl 45 tert-butyl
--CH.sub.2-- 15 46 tert-butyl bond 2-chloro-phenyl 47 tert-butyl
bond 4-chloro-phenyl 48 tert-butyl bond 3,4,5-trimethoxy- phenyl 49
tert-butyl bond 16 50 tert-butyl bond 17 51 tert-butyl
--O--CH.sub.2--, phenyl X attaches directly to the CH.sub.2
[0065] Still other preferred compounds of the present invention
include compounds 52-102 listed in Table II with formula I where A
is S as follows:
4TABLE II I 18 where A is S Compound R D X 52 methyl bond
4-bromophenyl 53 ethyl bond phenyl 54 n-propyl bond
3,4,5-trihydroxy- phenyl 55 i-propyl bond 3,4,5-trimethoxy- phenyl
56 n-butyl bond 3-hydroxyphenyl 57 t-butyl bond 4-nitro-naphthyl 58
s-butyl bond 3-hydroxy- naphthyl 59 pentyl bond benzyl 60 hexyl
bond 4-ethylphenyl 61 heptyl bond 4-ethenylphenyl 62 octyl bond
4-quinolyl 63 nonyl bond 2-thiazolyl 64 decyl bond 3-furyl 65
1,1,dimethylpropyl bond phenyl 66 ethenyl bond cyclohexyl 67
prop-2-enyl bond 3-bromocyclohexyl 68 phenyl bond adamantyl 69
naphthyl bond 4-indolyl 70 4-nitrophenyl bond 2-imidazolyl 71
4-hydroxyphenyl bond 1-naphthyl 72 4-chlorophenyl bond
4-nitrophenyl 73 4-methylphenyl bond 4-hydroxyphenyl 74
4-methoxyphenyl bond 3-piperidyl 75 4-dimethylamino- bond
3,4,5-trimethyl- phenyl phenyl 76 phenyl-ethyl-phenyl bond
3-pyridyl 77 4-nitro-3-hydroxy- bond 3,4,5-trifluoro- phenyl phenyl
78 1-pyridyl bond 1-pyrrolidyl 79 1-piperidyl bond 4-phenylazo-
phenyl 80 1-pyrrolidyl 2-bromo- 4-amino-3- propyl hydroxy-phenyl 81
cyclohexyl prop-2- 3,4,5-triamino- enyl phenyl 82 cyclopentyl
methyl 4-hydroxyphenyl 83 adamantyl ethyl phenyl 84 benzyl i-propyl
9-anthracenyl 85 4-hydroxybenzyl n-propyl 4-pyrenyl 86
3,4,5-trihydroxy- 2-imino- 3-furyl phenyl propyl 87 thiazolyl
2-thio- 3-thiophenyl propyl 88 2-phenylethyl 2-sulfonyl-
4-pyrimidinyl propyl 89 3-phenylpropyl ethenyl 4-isoquinolyl 90
2-phenylethenyl bond 4-sulfonylphenyl 91 3-phenylprop-2-enyl
chloro- 4-imino-phenyl methyl 92 3-bromophenyl
--CH.sub.2--N.dbd.CH-- 4-phenylethoxy- phenyl 93 4-fluoro-n-butyl
--CH.sub.2--S--CH.sub.2-- 4-ethylphenoxy- phenyl 94 3-methoxypropyl
--CH.sub.2--NH--CH.sub.2-- 4-phenoxy-phenyl 95 2-hydroxyethyl
--CH.sub.2--O--CH.sub.2-- 3-phenylpropyl- phenyl 96 tert-butyl
--CH.sub.2-- 19 97 tert-butyl bond 2-chloro-phenyl 98 tert-butyl
bond 4-chloro-phenyl 99 tert-butyl bond 3,4,5-trimethoxy- phenyl
100 tert-butyl bond 20 101 tert-butyl bond 21 102 tert-butyl
--O--CH.sub.2--, phenyl X attaches directly to the CH.sub.2
[0066] Another especially preferred embodiment of the invention is
a pharmaceutical composition which comprises (i) a therapeutically
effective amount of the compound of formula I; and (ii) a
pharmaceutically acceptable carrier.
[0067] As used herein, "alkyl" means a branched or unbranched
saturated hydrocarbon chain comprising a designated number of
carbon atoms. For example, C.sub.1-C.sub.6 straight or branched
alkyl hydrocarbon chain contains 1 to 6 carbon atoms, and includes
but is not limited to substituents such as methyl, ethyl, propyl,
iso-propyl, butyl, iso-butyl, tert-butyl, n-pentyl, n-hexyl, and
the like, unless otherwise indicated.
[0068] "Alkenyl" means a branched or unbranched unsaturated
hydrocarbon chain comprising a designated number of carbon atoms.
For example, C.sub.2-C.sub.6 straight or branched alkenyl
hydrocarbon chain contains 2 to 6 carbon atoms having at least one
double bond, and includes but is not limited to substituents such
as ethenyl, propenyl, isopropenyl, butenyl, iso-butenyl,
tert-butenyl, n-pentenyl, n-hexenyl, and the like, unless otherwise
indicated.
[0069] "Alkynyl" refers to a hydrocarbon chain containing a
carbon-carbon triple bond. For example, alkynyl groups include, but
are not limited to 1-butyne, 2-butyne, 2-pentyne,
3-methyl-1-butyne.
[0070] "Alkoxy", means the group --OR wherein R is alkyl as herein
defined. Preferably, R is a branched or unbranched saturated
hydrocarbon chain containing 1 to 6 carbon atoms.
[0071] "Cyclo", used herein as a prefix, refers to a structure
characterized by a closed ring.
[0072] "Halo" means at least one fluoro, chloro, bromo, or iodo
moiety, unless otherwise indicated.
[0073] "Amino" compounds include amine (NH.sub.2) as well as
substituted amino groups comprising alkyls of one through six
carbons.
[0074] "Ar" and "Aryl" as used herein refers to an aryl or
heteroaryl moiety which is substituted or unsubstituted, especially
a cyclic or fused cyclic ring and includes a mono-, bi-, or
tricyclic, alicylcic, carbocyclic or heterocyclic ring, wherein the
ring is either unsubstituted or substituted in one or more
position(s) with halo, haloalkyl, hydroxyl, nitro, trifluoromethyl,
C.sub.1-C.sub.6 straight or branched chain alkyl, C.sub.2-C.sub.6
straight or branched chain alkenyl, C.sub.1-C.sub.6 alkoxy,
C.sub.2-C.sub.6 alkenyloxy, phenoxy, benzyloxy, amino,
thiocarbonyl, ester, thioester, cyano, imino, alkylamino,
aminoalkyl, sulfhydryl, thioalkyl, and sulfonyl; wherein the
individual ring sizes are 5-8 members; wherein the heterocyclic
ring contains 1-4 heteroatom(s) selected from the group consisting
of O, N, or S; wherein aromatic or tertiary alkyl amines are
optionally oxidized to a corresponding N-oxide. Particularly
preferred aryl or heteroaryl moieties include but are not limited
to phenyl, benzyl, naphthyl, pyrrolyl, pyrrolidinyl, pyridinyl,
pyrimidinyl, purinyl, quinolinyl, isoquinolinyl, furyl,
tetrahydrofuryl, thiophenyl, imidazolyl, imidazolinyl, triazinyl,
morpholinyl, thiomorpholinyl, oxazolyl, isoxazolyl, triazolyl,
tetrahydrothienyl, benzopyranyl, benzofuranyl, benzothienyl,
indolyl, indolinyl, indolizinyl, cinnolinyl, phthalazinyl,
quinazolinyl, quinoxalinyl, pteridinyl, quinuclidinyl, carbazolyl,
acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl,
benzimidazolyl, benzthiazolyl, anthracenyl, thiazolyl,
isothiazolyl, pyrazinyl, piperazinyl, naphthyridinyl, pyrazolyl,
pyrazolidinyl, oxadiazolyl, thiadiazolyl, and thienyl.
[0075] "Aralkyl" as used herein refers to an aryl group that is
joined to a structure by one or more alkyl groups, e.g., benzyl,
phenethyl, and the like. Simiarly, "alkylaryl" refers to an alkyl
group that is joined to a structure by one or more aryl groups; and
"aryloxy" refers to an aryl group that is joined to a structure by
an oxygen group. Other similar combined terms may be interpreted
using the same formulation, such as: "arylamino" refers to an aryl
group that is joined to a structure by an amino group; or
"arylalkyloxy" refers to an aryl group that is joined to a
structure by an alkyloxy group, where the last named component,
oxygen, is directly joined to the structure.
[0076] "Carbocycle" as used herein refers to a cyclic carbon ring
structure, which may be saturated or unsaturated, and may also be
bridged or unbridged. For example, carbocycle moieties include, but
are not limited to, cyclohexyl, cyclopentyl, cycloheptyl,
cyclooctyl, adamantyl, and bicyclopentyl.
[0077] "Phenyl" includes all possible isomeric phenyl radicals,
optionally monosubstituted or multi-substituted with substituents
selected from the group consisting of amino, trifluoromethyl,
C.sub.1-C.sub.6 straight or branched chain alkyl, C.sub.2-C.sub.6
acceptable metabolites, and in the form of pharmaceutically
acceptable stereoisomers. These forms are all within the scope of
the invention. In practice, the use of these forms amounts to use
of the neutral compound.
[0078] "Pharmaceutically acceptable salt", "hydrate", "ester" or
"solvate" refers to a salt, hydrate, ester, or solvate of the
inventive compounds which possesses the desired pharmacological
activity and which is neither biologically nor otherwise
undesirable. Organic acids can be used to produce salts, hydrates,
esters, or solvates such as acetate, adipate, alginate, aspartate,
benzoate, benzenesulfonate, p-toluenesulfonate, bisulfate,
sulfamate, sulfate, naphthylate, butyrate, citrate, camphorate,
camphorsulfonate, cyclopentane-propionate, digluconate,
dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate,
glycerophosphate, hemisulfate heptanoate, hexanoate,
2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate,
2-naphthalenesulfonate, nicotinate, oxalate, tosylate and
undecanoate. Inorganic acids can be used to produce salts,
hydrates, esters, or solvates such as hydro-chloride, hydrobromide,
hydroiodide, and thiocyanate.
[0079] Examples of suitable base salts, hydrates, esters, or
solvates include hydroxides, carbonates, and bicarbonates of
ammonia, alkali metal salts such as sodium, lithium and potassium
salts, alkaline earth metal salts such as calcium and magnesium
salts, aluminum salts, and zinc salts.
[0080] Salts, hydrates, esters, or solvates may also be formed with
organic bases. Organic bases suitable for the formation of
pharmaceutically acceptable base addition salts, hydrates, esters,
or solvates of the compounds of the present invention include those
that are non-toxic and strong enough to form such salts, hydrates,
esters, or solvates. For purposes of illustration, the class of
such organic bases may include mono-, di-, and trialkylamines, such
as methylamine, dimethylamine, triethylamine and dicyclohexylamine;
mono-, di- or trihydroxyalkylamines, such as mono-, di-, and
triethanolamine; amino acids, such as arginine and lysine;
guanidine; N-methyl-glucosamine; N-methyl-glucamine; L-glutamine;
N-methyl-piperazine; morpholine; ethylenediamine;
N-benzyl-phenethylamine; (trihydroxy-methyl)aminoethane; and the
like. straight or branched chain alkenyl, carbonyl, thiocarbonyl,
ester, thioester, alkoxy, alkenoxy, cyano, nitro, imino,
alkylamino, aminoalkyl, sulfhydryl, thioalkyl, sulfonyl, hydroxy,
halo, haloalkyl, NR.sub.2 wherein R.sub.2 is selected from the
group consisting of hydrogen, (C.sub.1-C.sub.6)-straight or
branched chain alkyl, (C.sub.3-C.sub.6) straight or branched chain
alkenyl or alkynyl, and (C.sub.1-C.sub.4) bridging alkyl wherein
said bridging alkyl forms a heterocyclic ring starting with the
nitrogen of NR.sub.1 and ending with one of the carbon atoms of
said alkyl or alkenyl chain, and wherein said heterocyclic ring is
optionally fused to an Ar group.
[0081] The compounds of the present invention possess one or more
asymmetric center(s) and thus can be produced as mixtures (racemic
and non-racemic) of stereoisomers, or as individual enantiomers or
diastereomers. The individual stereoisomers may be obtained by
using an optically active starting material, by resolving a racemic
or non-racemic mixture of an intermediate at some appropriate stage
of the synthesis, or by resolution of the compound of formula (I).
It is understood that the individual stereoisomers as well as
mixtures (racemic and non-racemic) of stereoisomers are encompassed
by the scope of the present invention. The S-stereoisomer at atom 1
of formula I is most preferred due to its greater activity.
[0082] "Isomers" are different compounds that have the same
molecular formula and includes cyclic isomers such as (iso)indole
and other isomeric forms of cyclic moieties. "Stereoisomers" are
isomers that differ only in the way the atoms are arranged in
space. "Enantiomers" are a pair of stereoisomers that are
non-superimposable mirror images of each other. "Diastereoisomers"
are stereoisomers which are not mirror images of each other.
"Racemic mixture" means a mixture containing equal parts of
individual enantiomers. "Non-racemic mixture" is a mixture
containing unequal parts of individual enantiomers or
stereoisomers.
[0083] The compounds of the invention may be useful in a free base
form, in the form of pharmaceutically acceptable salts,
pharmaceutically acceptable hydrates, pharmaceutically acceptable
esters, pharmaceutically acceptable solvates, pharmaceutically
acceptable prodrugs, pharmaceutically See, for example,
"Pharmaceutical Salts," J. Pharm. Sci., 66:1, 1-19 (1977).
Accordingly, basic nitrogen-containing groups can be quaternized
with agents including: lower alkyl halides such as methyl, ethyl,
propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates
such as dimethyl, diethyl, dibutyl and diamyl sulfates; long chain
halides such as decyl, lauryl, myristyl and stearyl chlorides,
bromides and iodides; and aralkyl halides such as benzyl and
phenethyl bromides.
[0084] The acid addition salts, hydrates, esters, or solvates of
the basic compounds may be prepared either by dissolving the free
base of a PARP inhibitor in an aqueous or an aqueous alcohol
solution or other suitable solvent containing the appropriate acid
or base, and isolating the salt by evaporating the solution.
Alternatively, the free base of the PARP inhibitor may be reacted
with an acid, as well as reacting the PARP inhibitor having an acid
group thereon with a base, such that the reactions are in an
organic solvent, in which case the salt separates directly or can
be obtained by concentrating the solution.
[0085] "Pharmaceutically acceptable prodrug" refers to a derivative
of the inventive compounds which undergoes biotransformation prior
to exhibiting its pharmacological effect(s). The prodrug is
formulated with the objective(s) of improved chemical stability,
improved patient acceptance and compliance, improved
bioavailability, prolonged duration of action, improved organ
selectivity, improved formulation (e.g., increased
hydrosolubility), and/or decreased side effects (e.g., toxicity).
The prodrug can be readily prepared from the inventive compounds
using methods known in the art, such as those described by Burger's
Medicinal Chemistry and Drug Chemistry, Fifth Ed., Vol. 1, pp.
172-178, 949-982 (1995). For example, the inventive compounds can
be transformed into prodrugs by converting one or more of the
hydroxy or carboxy groups into esters.
[0086] "Pharmaceutically acceptable metabolite" refers to drugs
that have undergone a metabolic transformation. After entry into
the body, most drugs are substrates for chemical reactions that may
change their physical properties and biologic effects. These
metabolic conversions, which usually affect the polarity of the
compound, alter the way in which drugs are distributed in and
excreted from the body. However, in some cases, metabolism of a
drug is required for therapeutic effect. For example, anticancer
drugs of the antimetabolite class must be converted to their active
forms after they have been transported into a cancer cell. Since
must drugs undergo metabolic transformation of some kind, the
biochemical reactions that play a role in drug metabolism may be
numerous and diverse. The main site of drug metabolism is the
liver, although other tissues may also participate.
[0087] A feature characteristic of many of these transformations is
that the metabolic products are more polar than the parent drugs,
although a polar drug does sometimes yield a less polar product.
Substances with high lipid/water partition coefficients, which pass
easily across membranes, also diffuse back readily from tubular
urine through the renal tubular cells into the plasma. Thus, such
substances tend to have a low renal clearance and a long
persistence in the body. If a drug is metabolized to a more polar
compound, one with a lower partition coefficient, its tubular
reabsorption will be greatly reduced. Moreover, the specific
secretory mechanisms for anions and cations in the proximal renal
tubules and in the parenchymal liver cells operate upon highly
polar substances.
[0088] As a specific example, phenacetin (acetophenetidin) and
acetanilide are both mild analgesic and antipyretic agents, but are
each transformed within the body to a more polar and more effective
metabolite, p-hydroxyacetanilid (acetaminophen), which is widely
used today. When a dose of acetanilid is given to a person, the
successive metabolites peak and decay in the plasma sequentially.
During the first hour, acetanilid is the principal plasma
component. In the second hour, as the acetanilid level falls, the
metabolite acetaminophen concentration reaches a peak. Finally,
after a few hours, the principal plasma component is a further
metabolite that is inert and can be excreted from the body. Thus,
the plasma concentrations of one or more metabolites, as well as
the drug itself, can be pharmacologically important.
[0089] The reactions involved in drug metabolism are often
classified into two groups, as shown in the Table II. Phase I (or
functionalization) reactions generally consist of (1) oxidative and
reductive reactions that alter and create new functional groups and
(2) hydrolytic reactions that cleave esters and amides to release
masked functional groups. These changes are usually in the
direction of increased polarity.
[0090] Phase II reactions are conjugation reactions in which the
drug, or often a metabolite of the drug, is coupled to an
endogenous substrate, such as glucuronic acid, acetic acid, or
sulfuric acid.
5TABLE II Phase I Reactions (functionalization reactions): (1)
Oxidation via the hepatic microsomal P450 system: Aliphatic
oxidation Aromatic hydroxylation N-Dealkylation O-Dealkylation
S-Dealkylation Epoxidation Oxidative deamination Sulfoxide
formation Desulfuration N-Oxidation and N-hydroxylation
Dehalogenation (2) Oxidation via nonmicrosomal mechanisms: Alcohol
and aldehyde oxidation Purine oxidation Oxidative deamination
(monoamine oxidase and diamine oxidase) (3) Reduction: Azo and
nitro reduction (4) Hydrolysis: Ester and amide hydrolysis Peptide
bond hydrolysis Epoxide hydration Phase II Reactions (conjugation
reactions): (1) Glucuronidation (2) Acetylation (3) Mercacturic
acid formation (4) Sulfate conjugation (5) N-, O-, and
S-methylation (6) Trans-sulfuration
[0091] The compounds of the present invention exhibit
pharmacological activity and are, therefore, useful as
pharmaceuticals. In particular, the compounds exhibit central
nervous and cardiac vesicular system activity.
[0092] It is understood that tautomeric forms, when possible, are
included in the invention. For example, the tautomeric forms of the
following compounds are exemplary: 22
[0093] Typically, the novel compounds of the invention will have an
IC.sub.50 for inhibiting poly(ADP-ribose) synthetase in vitro of
100 .mu.M or lower, preferably 50 .mu.M or lower, more preferably
30 .mu.M or lower, more preferably 10 .mu.M or lower, and most
preferably 40 nM or lower.
[0094] There are multiple routes which may be undertaken to prepare
the ortho-diphenol compounds of the present invention using
commercially available materials and reagents. An example of one
such scheme is set forth below in Scheme I:
[0095] SCHEME I 23
[0096] where R is any substituent as described herein, including:
24
[0097] The compounds of the present invention may be synthesized
using the synthetic scheme shown in Scheme 1. The starting
materials and reagents required for the scheme 1 synthesis of the
compounds of the present invention may be obtained from one or more
of the following Chemical Supply Companies: Acros, Aldrich, Fluka,
JT-Baker, Maybridge, ICN, Lancaster, Sigma, Avocado, Coalite,
E-Merck, Eastern-Chemical, EM-Science, Kanto, Octel, and others. To
a solution of 4-tert-butylpyrocatechol (1.66 g, 0.01 M) in DMA (50
ml) a powdered t-BuOK (1.12 g, 0.01 M) was added in one portion at
room temperature under stirring. The formed solution was stirred
for 15 minutes, and the corresponding acid chloride (0.011 M) in
THF (10 ml) was added dropwise within 20-30 minutes upon continuous
stirring at room temperature. The mixture was stirred for an
additional 4-8 hours, and the content was poured into stirred
ice-water (100 g). After 3 hours of stirring, the formed solid
product was filtered and recrystallized from aqueous ethanol; oily
products were extracted with ethyl acetate (2.times.50 ml).
Extracts were thoroughly washed with water (5.times.50 ml),
separated, dried over MgSO.sub.4 anhydrous, and the solvents were
evaporated in vacuum.
[0098] Methods of Using the Compounds of the Invention
[0099] The ortho-diphenol compounds of the present invention can
treat or prevent tissue damage resulting from cell damage or death
due to necrosis or apoptosis; can ameliorate neural or
cardiovascular tissue damage, including that following focal
ischemia, myocardial infarction, and reperfusion injury; can treat
various diseases and conditions caused or exacerbated by PARP
activity; can extend or increase the lifespan or proliferative
capacity of cells; can alter the gene expression of senescent
cells; and can radiosensitize cells. Generally, inhibition of PARP
activity spares the cells from energy loss, preventing irreversible
depolarization of the neurons, and thus, provides neuroprotection.
While not being bound to any one particular theory, it is thought
that PARP activation may play a common role in still other
excitotoxic mechanisms, perhaps as yet undiscovered, in addition to
the production of free radicals and NO.
[0100] For the foregoing reasons, the present invention further
relates to a method of administering a therapeutically effective
amount of the above-identified compounds in an amount sufficient to
inhibit PARP activity, to treat or prevent tissue damage resulting
from cell damage or death due to necrosis or apoptosis, to effect a
neuronal activity not mediated by glutamate neurotoxicity, to
effect a neuronal activity mediated by glutamate neurotoxicity, to
treat neural tissue damage resulting from ischemia and reperfusion
injury, neurological disorders and neurodegenerative diseases; to
prevent or treat vascular stroke; to treat or prevent
cardiovascular disorders; to treat other conditions and/or
disorders such as age-related macular degeneration, AIDS and other
immune diseases, arthritis, atherosclerosis, cachexia, cancer,
degenerative diseases of skeletal muscle involving replicative
senescence, diabetes, head trauma, immune senescence, inflammatory
bowel disorders (such as colitis and Crohn's disease), muscular
dystrophy, osteoarthritis, osteoporosis, chronic and/or acute pain
(such as neuropathic pain), renal failure, retinal ischemia, septic
shock (such as endotoxic shock), and skin aging; to extend the
lifespan and proliferative capacity of cells; to alter gene
expression of senescent cells; or to radiosensitize hypoxic tumor
cells. The present invention also relates to treating diseases and
conditions in an animal which comprises administering to said
animal a therapeutically effective amount of the above-identified
compounds.
[0101] In particular, the present invention relates to a method of
treating, preventing or inhibiting a neurological disorder in an
animal, which comprises administering to said animal a
therapeutically effective amount of the above-identified compounds.
In a particularly preferred embodiment, the neurological disorder
is selected from the group consisting of peripheral neuropathy
caused by physical injury or disease state, traumatic brain injury,
physical damage to the spinal cord, stroke associated with brain
damage, focal ischemia, global ischemia, reperfusion injury,
demyelinating disease and neurological disorder relating to
neurodegeneration. Another preferred embodiment is when the
reperfusion injury is a vascular stroke. Yet another preferred
embodiment is when the peripheral neuropathy is caused by
Guillain-Barre syndrome. Still another preferred embodiment is when
the demyelinating disease is multiple sclerosis. Another preferred
embodiment is when the neurological disorder relating to
neurodegeneration is selected from the group consisting of
Alzheimer's Disease, Parkinson's Disease, and amyotrophic lateral
sclerosis.
[0102] Yet another preferred embodiment is a method of treating,
preventing or inhibiting a cardiovascular disease in an animal,
such as angina pectoris, myocardial infarction, cardiovascular
ischemia, and cardiovascular tissue damage related to PARP
activation, by administering to said animal an effective amount of
the compounds of the present invention.
[0103] The present invention also contemplates the use of compound
of Formula I for inhibiting PARP activity; and/or for treating,
preventing or inhibiting tissue damage resulting from cell damage
or death due to necrosis or apoptosis; and/or for treating,
preventing or inhibiting a neurological disorder in an animal.
[0104] In a particularly preferred embodiment, the neurological
disorder is selected from the group consisting of peripheral
neuropathy caused by physical injury or disease state, traumatic
brain injury, physical damage to the spinal cord, stroke associated
with brain damage, focal ischemia, global ischemia, reperfusion
injury, demyelinating disease and neurological disorder relating to
neurodegeneration.
[0105] Another preferred embodiment is when the reperfusion injury
is a vascular stroke. Yet another preferred embodiment is when the
peripheral neuropathy is caused by Guillain-Barre syndrome. Still
another preferred embodiment is when the demyelinating disease is
multiple sclerosis. Another preferred embodiment is when the
neurological disorder relating to neurodegeneration is selected
from the group consisting of Alzheimer's Disease, Parkinson's
Disease, and amyotrophic lateral sclerosis.
[0106] The present invention also contemplates the use of the
compound of formula I in the preparation of a medicament for the
treatment of any of the diseases and disorders in an animal
described herein.
[0107] In a particular embodiment, the disease or disorder is a
neurological disorder.
[0108] In a particularly preferred embodiment, the neurological
disorder is selected from the group consisting of peripheral
neuropathy caused by physical injury or disease state, traumatic
brain injury, physical damage to the spinal cord, stroke associated
with brain damage, focal ischemia, global ischemia, reperfusion
injury, demyelinating disease and neurological disorder relating to
neurodegeneration. Another preferred embodiment is when the
reperfusion injury is a vascular stroke. Yet another preferred
embodiment is when the peripheral neuropathy is caused by
Guillain-Barre syndrome.
[0109] Still another preferred embodiment is when the demyelinating
disease is multiple sclerosis. Another preferred embodiment is when
the neurological disorder relating to neurodegeneration is selected
from the group consisting of Alzheimer's Disease, Parkinson's
Disease, and amyotrophic lateral sclerosis.
[0110] The term "preventing neurodegeneration" includes the ability
to prevent neurodegeneration in patients newly diagnosed as having
a neurodegenerative disease, or at risk of developing a new
degenerative disease and for preventing further neurodegeneration
in patients who are already suffering from or have symptoms of a
neurodegenerative disease.
[0111] The term "treatment" as used herein covers any treatment of
a disease and/or condition in an animal, particularly a human, and
includes:
[0112] (i) preventing a disease and/or condition from occurring in
a subject which may be predisposed to the disease and/or condition
but has not yet been diagnosed as having it;
[0113] (ii) inhibiting the disease and/or condition, i.e.,
arresting its development; or
[0114] (iii) relieving the disease and/or condition, i.e., causing
regression of the disease and/or condition.
[0115] As used herein, the term "neural tissue damage resulting
from ischemia and reperfusion injury" includes neurotoxicity, such
as seen in vascular stroke and global and focal ischemia. As used
herein, the term "neurodegenerative diseases," includes Alzheimer's
disease, Parkinson's disease and Huntington's disease.
[0116] The term "ischemia" relates to localized tissue anemia due
to obstruction of the inflow of arterial blood. Global ischemia
occurs under conditions in which blood flow to the entire brain
ceases for a period of time, such as may result from cardiac
arrest. Focal ischemia occurs under conditions in which a portion
of the brain is deprived of its normal blood supply, such as may
result from thromboembolytic occlusion of a cerebral vessel,
traumatic head injury, edema, and brain tumors.
[0117] The term "cardiovascular disease" relates to myocardial
infarction, angina pectoris, vascular or myocardial ischemia, and
related conditions as would be known by those of skill in the art
which involve dysfunction of or tissue damage to the heart or
vasculature, and especially, but not limited to, tissue damage
related to PARP activation.
[0118] The term "radiosensitizer", as used herein, is defined as a
molecule, preferably a low molecular weight molecule, administered
to animals in therapeutically effective amounts to increase the
sensitivity of the cells to be radiosensitized to electromagnetic
radiation and/or to promote the treatment of diseases which are
treatable with electromagnetic radiation. Diseases which are
treatable with electromagnetic radiation include neoplastic
diseases, benign and malignant tumors, and cancerous cells.
Electromagnetic radiation treatment of other diseases not listed
herein are also contemplated by the present invention. The terms
"electromagnetic radiation" and "radiation" as used herein
includes, but is not limited to, radiation having the wavelength of
10.sup.-20 to 10.sup.0 meters. Preferred embodiments of the present
invention employ the electromagnetic radiation of: gamma-radiation
(10.sup.-20 to 10.sup.-13 m) x-ray radiation (10.sup.-11 to
10.sup.-9 m), ultraviolet light (10 nm to 400 nm), visible light
(400 nm to 700 nm), infrared radiation (700 nm to 1.0 mm), and
microwave radiation (1 mm to 30 cm).
[0119] Compositions and Methods for Effecting Neuronal Activity
[0120] Preferably, the compounds of the invention inhibit PARP
activity and, thus, are believed to be useful for treating neural
tissue damage, particularly damage resulting from cerebral ischemia
and reperfusion injury or neurodegenerative diseases in animals.
The term "nervous tissue" refers to the various components that
make up the nervous system including, without limitation, neurons,
neural support cells, glia, Schwann cells, vasculature contained
within and supplying these structures, the central nervous system,
the brain, the brain stem, the spinal cord, the junction of the
central nervous system with the peripheral nervous system, the
peripheral nervous system, and allied structures.
[0121] Further, according to the invention, an effective
therapeutic-amount of the compounds and compositions described
above are administered to animals to effect a neuronal activity,
particularly one that is not mediated by NMDA-receptor mediated
neurotoxicity. Such neuronal activity may consist of stimulation of
damaged neurons, promotion of neuronal regeneration, prevention of
neurodegeneration and treatment of a neurological disorder.
Accordingly, the present invention further relates to a method of
effecting a neuronal activity in an animal, comprising
administering an effective amount of the compound of formula I to
said animal.
[0122] Examples of neurological disorders that are treatable by the
method of using the present invention include, without limitation,
trigeminal neuralgia; glossopharyngeal neuralgia; Bell's Palsy;
myasthenia gravis; muscular dystrophy; amyotrophic lateral
sclerosis; progressive muscular atrophy; progressive bulbar
inherited muscular atrophy; herniated, ruptured or prolapsed
invertebrate disk syndromes; cervical spondylosis; plexus
disorders; thoracic outlet destruction syndromes; peripheral
neuropathies such as those caused by lead, dapsone, ticks,
porphyria, or Guillain-Barre syndrome; Alzheimer's disease;
Huntington's Disease and Parkinson's disease. The term
"neurodegenerative diseases" includes Alzheimer's disease,
Parkinson's disease and Huntington's disease.
[0123] The term "nervous insult" refers to any damage to nervous
tissue and any disability or death resulting therefrom. The cause
of nervous insult may be metabolic, toxic, neurotoxic, iatrogenic,
thermal or chemical, and includes without limitation, ischemia,
hypoxia, cerebrovascular accident, trauma, surgery, pressure, mass
effect, hemmorrhage, radiation, vasospasm, neurodegenerative
disease, infection, Parkinson's disease, amyotrophic lateral
sclerosis (ALS), myelination/demyelination process, epilepsy,
cognitive disorder, glutamate abnormality and secondary effects
thereof.
[0124] The term "neuroprotective" refers to the effect of reducing,
arresting or ameliorating nervous insult, and protecting,
resuscitating, or reviving nervous tissue that has suffered nervous
insult.
[0125] The term "preventing neurodegeneration" includes the ability
to prevent neurodegeneration in patients diagnosed as having a
neurodegenerative disease or who are at risk of developing a
neurodegenerative disease. The term also encompasses preventing
further neurodegeneration in patients who are already suffering
from or have symptoms of a neurodegenerative disease.
[0126] The term "treating" refers to:
[0127] (i) preventing a disease, disorder or condition from
occurring in an animal that may be predisposed to the disease,
disorder and/or condition, but has not yet been diagnosed as having
it;
[0128] (ii) inhibiting the disease, disorder or condition, i.e.,
arresting its development; and
[0129] (iii) relieving the disease, disorder or condition, i.e.,
causing regression of the disease, disorder and/or condition.
[0130] The method of the present invention is particularly useful
for treating a neurological disorder selected from the group
consisting of: peripheral neuropathy caused by physical injury or
disease state; head trauma, such as traumatic brain injury;
physical damage to the spinal cord; stroke associated with brain
damage, such as vascular stroke associated with hypoxia and brain
damage, focal cerebral ischemia, global cerebral ischemia, and
cerebral reperfusion injury; demyelinating diseases, such as
multiple sclerosis; and neurological disorders related to
neurodegeneration, such as Alzheimer's Disease, Parkinson's
Disease, Huntington's Disease and amyotrophic lateral sclerosis
(ALS).
[0131] The term "neural tissue damage resulting from ischemia and
reperfusion injury and neurodegenerative diseases" includes
neurotoxicity, such as seen in vascular stroke and global and focal
ischemia.
[0132] Treating Other PARP-Related Disorders
[0133] The compounds, compositions and methods of the present
invention are particularly useful for treating or preventing tissue
damage resulting from cell death or damage due to necrosis or
apoptosis.
[0134] The compounds, compositions and methods of the invention can
also be used to treat a cardiovascular disorder in an animal, by
administering an effective amount of the compound of formula to the
animal. As used herein, the term "cardiovascular disorders" refers
to those disorders that can either cause ischemia or are caused by
reperfusion of the heart. Examples include, but are not limited to,
coronary artery disease, angina pectoris, myocardial infarction,
cardiovascular tissue damage caused by cardiac arrest,
cardiovascular tissue damage caused by cardiac bypass, cardiogenic
shock, and related conditions that would be known by those of
ordinary skill in the art or which involve dysfunction of or tissue
damage to the heart or vasculature, especially, but not limited to,
tissue damage related to PARP activation.
[0135] For example, the methods of the invention are believed to be
useful for treating cardiac tissue damage, particularly damage
resulting from cardiac ischemia or caused by reperfusion injury in
animals. The methods of the invention are particularly useful for
treating cardiovascular disorders selected from the group
consisting of: coronary artery disease, such as atherosclerosis;
angina pectoris; myocardial infarction; myocardial ischemia and
cardiac arrest; cardiac bypass; and cardiogenic shock. The methods
of the invention are particularly helpful in treating the acute
forms of the above cardiovascular disorders.
[0136] Further, the methods of the invention can be used to treat
tissue damage resulting from cell damage or death due to necrosis
or apoptosis, neural tissue damage resulting from ischemia and
reperfusion injury, neurological disorders and neurodegenerative
diseases; to prevent or treat vascular stroke; to treat or prevent
cardiovascular disorders; to treat other conditions and/or
disorders such as age-related macular degeneration, AIDS and other
immune diseases, arthritis, atherosclerosis, cachexia, cancer,
degenerative diseases of skeletal muscle involving replicative
senescence, diabetes, head trauma, immune senescence, inflammatory
bowel disorders (such as colitis and Crohn's disease), muscular
dystrophy, osteoarthritis, osteoporosis, chronic and/or acute pain
(such as neuropathic pain), renal failure, retinal ischemia, septic
shock (such as endotoxic shock), and skin aging; to extend the
lifespan and proliferative capacity of cells; to alter gene
expression of senescent cells; or to radiosensitize tumor cells
[0137] Further still, the methods of the invention can be used to
treat cancer and to radiosensitize tumor cells. The term "cancer"
is interpreted broadly. The compounds of the present invention can
be "anti-cancer agents", which term also encompasses "anti-tumor
cell growth agents" and "anti-neoplastic agents". For example, the
methods of the invention are useful for treating cancers and
radiosensitizing tumor cells in cancers such as ACTH-producing
tumors, acute lymphocytic leukemia, acute nonlymphocytic leukemia,
cancer of the adrenal cortex, bladder cancer, brain cancer, breast
cancer, cervical cancer, chronic lymphocytic leukemia, chronic
myelocytic leukemia, colorectal cancer, cutaneous T-cell lymphoma,
endometrial cancer, esophageal cancer, Ewing's sarcoma, gallbladder
cancer, hairy cell leukemia, head & neck cancer, Hodgkin's
lymphoma, Kaposi's sarcoma, kidney cancer, liver cancer, lung
cancer (small and/or non-small cell), malignant peritoneal
effusion, malignant pleural effusion, melanoma, mesothelioma,
multiple myeloma, neuroblastoma, non-Hodgkin's lymphoma,
osteosarcoma, ovarian cancer, ovary (germ cell) cancer, prostate
cancer, pancreatic cancer, penile cancer, retinoblastoma, skin
cancer, soft-tissue sarcoma, squamous cell carcinomas, stomach
cancer, testicular cancer, thyroid cancer, trophoblastic neoplasms,
uterine cancer, vaginal cancer, cancer of the vulva and Wilm's
tumor.
[0138] The term "radiosensitizer", as used herein, is defined as a
molecule, preferably a low molecular weight molecule, administered
to animals in therapeutically effective amounts to increase the
sensitivity of the cells to be radiosensitized to electromagnetic
radiation and/or to promote the treatment of diseases which are
treatable with electromagnetic radiation. Diseases which are
treatable with electromagnetic radiation include neoplastic
diseases, benign and malignant tumors, and cancerous cells.
Electromagnetic radiation treatment of other diseases not listed
herein are also contemplated by the present invention. The terms
"electromagnetic radiation" and "radiation" as used herein
includes, but is not limited to, radiation having the wavelength of
10.sup.-20 to 10.sup.0 meters. Preferred embodiments of the present
invention employ the electromagnetic radiation of: gamma-radiation
(10.sup.-20 to 10.sup.-13 m) x-ray radiation (10.sup.-11 to
10.sup.-9 m), ultraviolet light (10 nm to 400 nm), visible light
(400 nm to 700 nm), infrared radiation (700 nm to 1.0 mm), and
microwave radiation (1 mm to 30 cm).
[0139] Radiosensitizers are known to increase the sensitivity of
cancerous cells to the toxic effects of electromagnetic radiation.
Several mechanisms for the mode of action of radiosensitizers have
been suggested in the literature including: hypoxic cell
radiosensitizers (e.g., 2-nitro-imidazole compounds, and
benzotriazine dioxide compounds) promote the reoxygenation of
hypoxic tissue and/or catalyze the generation of damaging oxygen
radicals; non-hypoxic cell radiosensitizers (e.g., halogenated
pyrimidines) can be analogs of DNA bases and preferentially
incorporate into the DNA of cancer cells and thereby promote the
radiation-induced breaking of DNA molecules and/or prevent the
normal DNA repair mechanisms; and various other potential
mechanisms of action have been hypothesized for radiosensitizers in
the treatment of disease.
[0140] Many cancer treatment protocols currently employ
radiosensitizers activated by the electromagnetic radiation of
x-rays. Examples of x-ray activated radiosensitizers include, but
are not limited to, the following: metronidazole, misonidazole,
desmethylmisonidazole, pimonidazole, etanidazole, nimorazole,
mitomycin C, RSU 1069, SR 4233, EO 9, RB 6145, nicotinamide,
5-bromodeoxyuridine (BUdR), 5-iododeoxyuridine (IUdR),
bromodeoxycytidine, fluorodeoxyuridine (FudR), hydroxyurea,
cisplatin, and therapeutically effective analogs and derivatives of
the same.
[0141] Photodynamic therapy (PDT) of cancers employs visible light
as the radiation activator of the sensitizing agent. Examples of
photodynamic radiosensitizers include the following, but are not
limited to: hematoporphyrin derivatives, Photofrin, benzoporphyrin
derivatives, NPe6, tin etioporphyrin SnET2, pheoborbide-a,
bacteriochlorophyll-a, naphthalocyanines, phthalocyanines, zinc
phthalocyanine, and therapeutically effective analogs and
derivatives of the same.
[0142] Radiosensitizers may be administered in conjunction with a
therapeutically effective amount of one or more other compounds,
including but not limited to: compounds which promote the
incorporation of radiosensitizers to the target cells; compounds
which control the flow of therapeutics, nutrients, and/or oxygen to
the target cells; chemotherapeutic agents which act on the tumor
with or without additional radiation; or other therapeutically
effective compounds for treating cancer or other disease. Examples
of additional therapeutic agents that may be used in conjunction
with radiosensitizers include, but are not limited to:
5-fluorouracil, leucovorin, 5'-amino-5'deoxythymidine, oxygen,
carbogen, red cell transfusions, perfluorocarbons (e.g.,
Fluosol-DA), 2,3-DPG, BW12C, calcium channel blockers,
pentoxyfylline, antiangiogenesis compounds, hydralazine, and L-BSO.
Examples of chemotherapeutic agents that may be used in conjunction
with radiosensitizers include, but are not limited to: adriamycin,
camptothecin, carboplatin, cisplatin, daunorubicin, docetaxel,
doxorubicin, interferon (alpha, beta, gamma), interleukin 2,
irinotecan, paclitaxel, topotecan, and therapeutically effective
analogs and derivatives of the same.
[0143] The compounds of the present invention may also be used for
radiosensitizing tumor cells.
[0144] The term "treating" refers to:
[0145] (i) preventing a disease, disorder or condition from
occurring in an animal that may be predisposed to the disease,
disorder and/or condition, but has not yet been diagnosed as having
it;
[0146] (ii) inhibiting the disease, disorder or condition, i.e.,
arresting its development; and
[0147] (iii) relieving the disease, disorder or condition, i.e.,
causing regression of the disease, disorder and/or condition.
[0148] Pharmaceutical Compositions of the Invention
[0149] The present invention also relates to a pharmaceutical
composition comprising (i) a therapeutically effective amount of
the compound of formula I and (ii) a pharmaceutically acceptable
carrier.
[0150] The above discussion relating to the preferred embodiments'
utility and administration of the compounds of the present
invention also applies to the pharmaceutical composition of the
present invention.
[0151] The term "pharmaceutically acceptable carrier" as used
herein refers to any carrier, diluent, excipient, suspending agent,
lubricating agent, adjuvant, vehicle, delivery system, emulsifier,
disintegrant, absorbent, preservative, surfactant, colorant,
flavorant, or sweetener.
[0152] For these purposes, the composition of the invention may be
administered orally, parenterally, by inhalation spray, topically,
rectally, nasally, bucally, vaginally, intraventricularly, via an
implanted reservoir in dosage formulations containing conventional
non-toxic pharmaceutically-acceptable carriers, or by any other
convenient dosage form. The term parenteral as used herein includes
subcutaneous, intravenous, intramuscular, intraperitoneal,
intrathecal, intraventricular, intrasternal, and intracranial
injection or infusion techniques.
[0153] When administered parenterally, the composition will
normally be in a unit dosage, sterile injectable form (solution,
suspension or emulsion) which is preferably isotonic with the blood
of the recipient with a pharmaceutically acceptable carrier.
Examples of such sterile injectable forms are sterile injectable
aqueous or oleaginous suspensions. These suspensions may be
formulated according to techniques known in the art using suitable
dispersing or wetting agents and suspending agents. The sterile
injectable forms may also be sterile injectable solutions or
suspensions in non-toxic parenterally-acceptable diluents or
solvents, for example, as solutions in 1,3-butanediol. Among the
acceptable vehicles and solvents that may be employed are water,
saline, Ringer's solution, dextrose solution, isotonic sodium
chloride solution, and Hanks' solution. In addition, sterile, fixed
oils are conventionally employed as solvents or suspending mediums.
For this purpose, any bland fixed oil may be employed including
synthetic mono- or di-glycerides, corn, cottonseed, peanut, and
sesame oil. Fatty acids such as ethyl oleate, isopropyl myristate,
and oleic acid and its glyceride derivatives, including olive oil
and castor oil, especially in their polyoxyethylated versions, are
useful in the preparation of injectables. These oil solutions or
suspensions may also contain long-chain alcohol diluents or
dispersants.
[0154] Sterile saline is a preferred carrier, and the compounds are
often sufficiently water soluble to be made up as a solution for
all foreseeable needs. The carrier may contain minor amounts of
additives, such as substances that enhance solubility, isotonicity,
and chemical stability, e.g., anti-oxidants, buffers and
preservatives.
[0155] Formulations suitable for nasal or buccal administration
(such as self-propelling powder dispensing formulations) may
comprise about 0.1% to about 5% w/w, for example 1% w/w of active
ingredient. The formulations for human medical use of the present
invention comprise an active ingredient in association with a
pharmaceutically acceptable carrier therefore and optionally other
therapeutic ingredient(s).
[0156] When administered orally, the composition will usually be
formulated into unit dosage forms such as tablets, cachets, powder,
granules, beads, chewable lozenges, capsules, liquids, aqueous
suspensions or solutions, or similar dosage forms, using
conventional equipment and techniques known in the art. Such
formulations typically include a solid, semisolid, or liquid
carrier. Exemplary carriers include lactose, dextrose, sucrose,
sorbitol, mannitol, starches, gum acacia, calcium phosphate,
mineral oil, cocoa butter, oil of theobroma, alginates, tragacanth,
gelatin, syrup, methyl cellulose, polyoxyethylene sorbitan
monolaurate, methyl hydroxybenzoate, propyl hydroxybenzoate, talc,
magnesium stearate, and the like.
[0157] The composition of the invention is preferably administered
as a capsule or tablet containing a single or divided dose of the
inhibitor. Preferably, the composition is administered as a sterile
solution, suspension, or emulsion, in a single or divided dose.
Tablets may contain carriers such as lactose and corn starch,
and/or lubricating agents such as magnesium stearate. Capsules may
contain diluents including lactose and dried corn starch.
[0158] A tablet may be made by compressing or molding the active
ingredient optionally with one or more accessory ingredients.
Compressed tablets may be 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, lubricant,
inert diluent, surface active, or dispersing agent. Molded tablets
may be made by molding in a suitable machine, a mixture of the
powdered active ingredient and a suitable carrier moistened with an
inert liquid diluent.
[0159] The compounds of this invention may also be administered
rectally in the form of suppositories. These compositions can be
prepared by mixing the drug with a suitable non-irritating
excipient which is solid at room temperature, but liquid at rectal
temperature, and, therefore, will melt in the rectum to release the
drug. Such materials include cocoa butter, beeswax, and
polyethylene glycols.
[0160] Compositions and methods of the invention also may utilize
controlled release technology. Thus, for example, the inventive
compounds may be incorporated into a hydrophobic polymer matrix for
controlled release over a period of days. The composition of the
invention may then be molded into a solid implant suitable for
providing efficacious concentrations of the PARP inhibitors over a
prolonged period of time without the need for frequent re-dosing.
Such controlled release films are well known to the art.
Particularly preferred are transdermal delivery systems. Other
examples of polymers commonly employed for this purpose that may be
used in the present-invention include nondegradable ethylene-vinyl
acetate copolymer an degradable lactic acid-glycolic acid
copolymers which may be used externally or internally. Certain
hydrogels such as poly(hydroxyethylmethacrylate) or
poly(vinylalcohol) also may be useful, but for shorter release
cycles than the other polymer release systems, such as those
mentioned above.
[0161] In a preferred embodiment, the carrier is a solid
biodegradable polymer or mixture of biodegradable polymers with
appropriate time release characteristics and release kinetics. The
composition of the invention may then be molded into a solid
implant suitable for providing efficacious concentrations of the
compounds of the invention over a prolonged period of time without
the need for frequent re-dosing. The composition of the present
invention can be incorporated into the biodegradable polymer or
polymer mixture in any suitable manner known to one of ordinary
skill in the art and may form a homogeneous matrix with the
biodegradable polymer, or may be encapsulated in some way within
the polymer, or may be molded into a solid implant.
[0162] In one embodiment, the biodegradable polymer or polymer
mixture is used to form a soft "depot" containing the
pharmaceutical composition of the present invention that can be
administered as a flowable liquid, for example, by injection, but
which remains sufficiently viscous to maintain the pharmaceutical
composition within the localized area around the injection site.
The degradation time of the depot so formed can be varied from
several days to a few years, depending upon the polymer selected
and its molecular weight. By using a polymer composition in
injectable form, even the need to make an incision may be
eliminated. In any event, a flexible or flowable delivery "depot"
will adjust to the shape of the space it occupies with the body
with a minimum of trauma to surrounding tissues. The pharmaceutical
composition of the present invention is used in amounts that are
therapeutically effective, and may depend upon the desired release
profile, the concentration of the pharmaceutical composition
required for the sensitizing effect, and the length of time that
the pharmaceutical composition has to be released for
treatment.
[0163] The PARP inhibitors are used in the composition in amounts
that are therapeutically effective. Said compositions may be
sterilized and/or contain adjuvants, such as preserving,
stabilizing, welling, or emulsifying agents, solution promoters,
salts for regulating the osmotic pressure, and/or buffers. In
addition, they may also contain other therapeutically valuable
substances. Said compositions are prepared according to
conventional mixing, granulating, or coating methods, and contain
about 0.1 to 75%, preferably about 1 to 50%, of the active
ingredient.
[0164] To be effective therapeutically as central nervous system
targets, the compounds of the present invention should readily
penetrate the blood-brain barrier when peripherally administered.
Compounds which cannot penetrate the blood-brain barrier can be
effectively administered by an intraventricular route or other
appropriate delivery system suitable for administration to the
brain.
[0165] Doses of the compounds preferably include pharmaceutical
dosage units comprising an efficacious quantity of active compound.
By an efficacious quantity is meant a quantity sufficient to
inhibit PARP and derive the beneficial effects therefrom through
administration of one or more of the pharmaceutical dosage units.
Preferably, the dose is sufficient to prevent or reduce the effects
of vascular stroke or other neurodegenerative diseases.
[0166] For medical use, the amount required of the active
ingredient to achieve a therapeutic effect will vary with the
particular compound, the route of administration, the mammal under
treatment, and the particular disorder or disease being treated. A
suitable systematic dose of a compound of the present invention or
a pharmacologically acceptable salt thereof for a mammal suffering
from, or likely to suffer from, any of condition as described
hereinbefore is in the range of about 0.01 mg/kg to about 100 mg/kg
of the active ingredient compound, the most preferred dosage being
about 0.1 to about 10 mg/kg.
[0167] It is understood, however, that a specific dose level for
any particular patient will depend upon a variety of factors
including the activity of the specific compound employed, the
molecular weight of the specific compound employed, the age, body
weight, general health, sex, diet, time of administration, rate of
excretion, drug combination, and the severity of the particular
disease being treated and form of administration.
[0168] It is understood that the ordinarily skilled physician or
veterinarian will readily determine and prescribe the effective
amount of the compound for prophylactic or therapeutic treatment of
the condition for which treatment is administered. In so
proceeding, the physician or veterinarian could employ an
intravenous bolus followed by an intravenous infusion and repeated
administrations, parenterally or orally, as considered appropriate.
While it is possible for an active ingredient to be administered
alone, it is preferable to present it as a formulation.
[0169] When preparing dosage form incorporating the compositions of
the invention, the compounds may also be blended with conventional
excipients such as binders, including gelatin, pregelatinized
starch, and the like; lubricants, such as hydrogenated vegetable
oil, stearic acid, and the like; diluents, such as lactose,
mannose, and sucrose; disintegrants, such as carboxymethylcellulose
and sodium starch glycolate; suspending agents, such as povidone,
polyvinyl alcohol, and the like; absorbants, such as silicon
dioxide; preservatives, such as methylparaben, propylparaben, and
sodium benzoate; surfactants, such as sodium lauryl sulfate,
polysorbate 80, and the like; colorants such as F.D.& C. dyes
and lakes; flavorants; and sweeteners.
[0170] The present invention relates to the use of the compounds of
formula I in the preparation of a medicament for the treatment of
any disease or disorder in an animal described herein.
[0171] PARP Assay
[0172] A convenient method to determine IC.sub.50 of a PARP
inhibitor compound is a PARP assay using purified recombinant human
PARP from Trevigan (Gaithersburg, Md.), as follows: The PARP enzyme
assay is set up on ice in a volume of 100 microliters consisting of
100 mM Tris-HCl (pH 8.0), 1 mM MgCl.sub.2, 28 mM KCl, 28 mM NaCl,
0.1 mg/ml of herring sperm DNA (activated as a 1 mg/ml stock for 10
minutes in a 0.15% hydrogen peroxide solution), 3.0 micromolar [3H]
nicotinamide adenine dinucleotide (470 mci/mmole), 7 micrograms/ml
PARP enzyme, and various concentrations of the compounds to be
tested. The reaction is initiated by incubating the mixture at
25.degree. C. After 15 minutes of incubation, the reaction is
terminated by adding 500 microliters of ice cold 20% (w/v)
trichloroacetic acid. The precipitate formed is transferred onto a
glass fiber filter (Packard Unifilter-GF/B) and washed three times
with ethanol. After the filter is dried, the radioactivity is
determined by scintillation counting. The compounds of this
invention were found to have potent enzymatic activity in the range
of a few nM to 20 M in IC.sub.50 in this inhibition assay.
[0173] Focal cerebral ischemia experiments were performed using
male Wistar rats weighing 250-300 g which were anesthetized with 4%
halothane. This anesthesia was maintained with 1.0-1.5% halothane
until the end of the surgery. The animals were placed in a warm
environment to avoid a decrease of body temperature during surgery.
An anterior midline cervical incision was made. The right common
carotid artery (CCA) was exposed and was isolated from the vagus
nerve. A silk suture was placed and tied around the CCA in
proximity to the heart. The external carotid artery (ECA) was then
exposed and was ligated with a silk suture. A puncture was made in
the CCA and a small catheter (PE 10, Ulrich & Co., St-Gallen,
Switzerland) was gently advanced to the lumen of the internal
carotid artery (ICA). The pterygopalatine artery was not occluded.
The catheter was tied in place with a silk suture. Then, a 4-0
nylon suture (Braun Medical, Crissier, Switzerland) was introduced
into the catheter lumen and was pushed until the tip blocked the
anterior cerebral artery. The length of catheter advanced into the
ICA was approximately 19 mm from the origin of the ECA. The suture
was maintained in this position by occlusion of the catheter by
heat. One cm of catheter and nylon suture were left protruding so
that the suture could be withdrawn to allow reperfusion. The skin
incision was then closed with wound clips and the animals
maintained in a warm environment during recovery from anesthesia.
Two hours later, the animals were re-anesthized, the clips were
discarded and the wound re-opened. The catheter was cut and the
suture was pulled out. The catheter was then obturated again by
heat, and wound clips were placed on the wound. The animals were
allowed to survive for 24 hours with free access to food and water.
The rats were sacrificed with CO.sub.2 and were decapitated. The
brains were immediately removed, frozen on dry ice and stored at
-80.degree. C. The brains were then cut in 0.02 mm-thick sections
in a cryocut at -19.degree. C., taking one of every 20 sections.
The sections were stained with cresyl violet according to the Nissl
procedure. Each section was examined under a light microscope and
the regional infarct area was determined according to the presence
of cells with morphological changes. Various doses of compounds
were tested in this model. The compounds were given in either
single or multiple doses, i.p. or i.v., at different times before
or after the onset of ischemia. Compounds of this invention were
found to have protection in the range of 20 to 80 per cent in this
assay.
[0174] The experiments of the heart ischemia/reperfusion injury
model were performed using female Sprague-Dawley rats weighing
300-350g which were anesthetized with intraperitoneal ketamine at a
dose of 150 mg/kg. The rats were endotracheally incubated and
ventilated with oxygen-enriched room air using a Harvard rodent
ventilator. Polyethylene catheters inserted into the carotid artery
and the femoral vein were used for artery blood ressure monitoring
and fluid administration, respectively. rterial pCO.sub.2 was
maintained between 35 and 45 mm Hg by adjusting the respiratory
rate. The rat chests were opened by median sternotomy, the
pericardium was incised, and the hearts were cradled with a latex
membrane tent. Hemodynamic data were obtained at baseline after at
least 15 minute stabilization from the end of the surgical
operation. The LAD (left anterior descending) coronary artery was
ligated for 40 minutes and was followed by 120 minutes of
reperfusion. After 120 minutes of reperfusion, the LAD artery was
reoccluded, and a 0.1 ml bolus of monastral blue dye was injected
into the left atrium to determine the ischemic risk region. The
hearts were then arrested with potassium chloride. The hearts were
cut into five 2-3 mm thick transverse slices, and each slice was
weighed. The sections were incubated in a 1% solution of
triphenyltetrazolium chloride to visualize the infarcted myocardium
located within the risk region. Infarct size was calculated by
summing the values for each left ventricular slice and expressed as
a fraction of the risk region of the left ventricle. Various doses
of compounds were tested in this model. The compounds were given in
either single or multiple doses, i.p or i.v., at different times
before or after the onset of ischemia. The compounds of this
invention were found to have ischemia/reperfusion injury protection
in the range of 10 to 40 percent in this assay.
[0175] As a result of their demonstrated PARP inhibition, the
compounds of this invention protect against ischemia-induced
degeneration of rat hippocampal neurons in vitro and thus may be
useful in disorders arising from cerebral ischemia such as stroke,
septic shock, or CNS degenerative disorders. They may also be
useful in protecting the spinal cord following trauma. As an
experimental result of ischemia/reperfusion injury in rats, the
present invention is further directed to a method of prophylactic
or therapeutic treatment of heart attack, cardiac arrest, cardiac
bypass, diabetes, or risk of damage which comprises administering
an effective amount of a compound of the present invention for PARP
inhibition in unit dosage form.
EXAMPLES
[0176] Further understanding of the present invention may be made
by reference to the following examples:
Example 1
[0177] 25
[0178] where R is any substituent as described herein, including:
26
[0179] The compounds of the present invention may be synthesized
using the synthetic scheme shown in Example 1. The starting
materials and reagents required for the synthesis of the compounds
of the present invention may be obtained from one or more of the
following Chemical Supply Companies: Acros, Aldrich, Fluka,
JT-Baker, Maybridge, ICN, Lancaster, Sigma, Avocado, Coalite,
E-Merck, Eastern-Chemical, EM-Science, Kanto, Octel, and others. To
a solution of 4-tert-butylpyrocatechol (1.66 g, 0.01 M) in DMA (50
ml) a powdered t-BuOK (1.12 g, 0.01 M) was added in one portion at
room temperature under stirring. The formed solution was stirred
for 15 minutes, and the corresponding acid chloride (0.011 M) in
THF (10 ml) was added dropwise within 20-30 minutes upon continuous
stirring at room temperature. The mixture was stirred for an
additional 4-8 hours, and the content was poured into stirred
ice-water (100 g). After 3 hours of stirring, the formed solid
product was filtered and recrystallized from aqueous ethanol; oily
products were extracted with ethyl acetate (2.times.50 ml).
Extracts were thoroughly washed with water (5.times.50 ml),
separated, dried over MgSO.sub.4 anhydrous, and the solvents were
evaporated in vacuum.
Example 2
[0180] Preparation of Other Ortho-Diphenol Compounds
[0181] Using the synthesis schemes described above and the methods
of the preceding examples, the following compounds of formula I are
synthesized:
6 27 Compound R D X 1 methyl bond 4-bromophenyl 2 ethyl bond phenyl
3 n-propyl bond 3,4,5-trihydroxy-phenyl 4 i-propyl bond
3,4,5-trimethoxy-phenyl 5 n-butyl bond 3-hydroxyphenyl 6 t-butyl
bond 4-nitro-naphthyl 7 s-butyl bond 3-hydroxy-naphthyl 8 pentyl
bond benzyl 9 hexyl bond 4-ethylphenyl 10 heptyl bond
4-ethenylphenyl 11 octyl bond 4-quinolyl 12 nonyl bond 2-thiazolyl
13 decyl bond 3-furyl 14 1,1,dimethylpropyl bond phenyl 15 ethenyl
bond cyclohexyl 16 prop-2-enyl bond 3-bromocyclohexyl 17 phenyl
bond adamantyl 18 naphthyl bond 4-indolyl 19 4-nitrophenyl bond
2-imidazolyl 20 4-hydroxyphenyl bond 1-naphthyl 21 4-chlorophenyl
bond 4-nitrophenyl 22 4-methylphenyl bond 4-hydroxyphenyl 23
4-methoxyphenyl bond 3-piperidyl 24 4-dimethyl-amino-phenyl bond
3,4,5-trimethyl-phenyl 25 phenyl-ethyl-phenyl bond 3-pyridyl 26
4-nitro-3-hydroxy-phenyl bond 3,4,5-trifluoro-phenyl 27 1-pyridyl
bond 1-pyrrolidyl 28 1-piperidyl bond 4-phenylazo-phenyl 29
1-pyrrolidyl 2-bromo-propyl 4-amino-3-hydroxy-phenyl 30 cyclohexyl
prop-2-enyl 3,4,5-triamino-phenyl 31 cyclopentyl methyl
4-hydroxyphenyl 32 adamantyl ethyl phenyl 33 benzyl i-propyl
9-anthracenyl 34 4-hydroxybenzyl n-propyl 4-pyrenyl 35
3,4,5-trihydroxy-phenyl 2-imino-propyl 3-furyl 36 thiazolyl
2-thio-propyl 3-thiophenyl 37 2-phenylethyl 2-sulfonyl-propyl
4-pyrimidinyl 38 3-phenylpropyl ethenyl 4-isoquinolyl 39
2-phenylethenyl bond 4-sulfonylphenyl 40 3-phenylprop-2-enyl
chloro-methyl 4-imino-phenyl 41 3-bromopropyl
--CH.sub.2--N.dbd.CH-- 4-phenylethoxy-phenyl 42 4-fluoro-n-butyl
--CH.sub.2--S--CH.sub.2-- 4-ethylphenoxy-phenyl 43 3-methoxypropyl
--CH.sub.2--NH--CH.sub.2-- 4-phenoxy-phenyl 44 2-hydroxyethyl
--CH.sub.2--O--CH.sub.2-- 3-phenylpropyl-phenyl 45 tert-butyl
--CH.sub.2-- 28 46 tert-butyl bond 2-chloro-phenyl 47 tert-butyl
bond 4-chloro-phenyl 48 tert-butyl bond 3,4,5-trimethoxy-phenyl 49
tert-butyl bond 29 50 tert-butyl bond 30 51 tert-butyl
--O--CH.sub.2--, X phenyl attaches directly to the CH.sub.2
Example 3
[0182] Preparation of Other Ortho-Diphenol Compounds
[0183] Using the synthesis schemes described above and the methods
of the preceding examples, the following compounds of formula I
where A is S are synthesized:
7 31 where A is S Compound R D X 52 methyl bond 4-bromophenyl 53
ethyl bond phenyl 54 n-propyl bond 3,4,5-trihydroxy-phenyl 55
i-propyl bond 3,4,5-trimethoxy-phenyl 56 n-butyl bond
3-hydroxyphenyl 57 t-butyl bond 4-nitro-naphthyl 58 s-butyl bond
3-hydroxy-naphthyl 59 pentyl bond benzyl 60 hexyl bond
4-ethylphenyl 61 heptyl bond 4-ethenylphenyl 62 octyl bond
4-quinolyl 63 nonyl bond 2-thiazolyl 64 decyl bond 3-furyl 65
1,1dimethylpropyl bond phenyl 66 ethenyl bond cyclohexyl 67
prop-2-enyl bond 3-bromocyclohexyl 68 phenyl bond adamantyl 69
naphthyl bond 4-indolyl 70 4-nitrophenyl bond 2-imidazolyl 71
4-hydroxyphenyl bond 1-naphthyl 72 4-chlorophenyl bond
4-nitrophenyl 73 4-methylphenyl bond 4-hydroxyphenyl 74
4-methoxyphenyl bond 3-piperidyl 75 4-dimethylamino-phenyl bond
3,4,5-trimethyl-phenyl 76 phenyl-ethyl-phenyl bond 3-pyridyl 77
4-nitro-3-hydroxy-phenyl bond 3,4,5-trifluoro-phenyl 78 1-pyridyl
bond 1-pyrrolidyl 79 1-piperidyl bond 4-phenylazo-phenyl 80
1-pyrrolidyl 2-bromo-propyl 4-amino-3-hydroxy-phenyl 81 cyclohexyl
prop-2-enyl 3,4,5-triamino-phenyl 82 cyclopentyl methyl
4-hydroxyphenyl 83 adamantyl ethyl phenyl 84 benzyl i-propyl
9-anthracenyl 85 4-hydroxybenzyl n-propyl 4-pyrenyl 86
3,4,5-trihydroxy-phenyl 2-imino-propyl 3-furyl 87 thiazolyl
2-thio-propyl 3-thiophenyl 88 2-phenylethyl 2-sulfonyl-propyl
4-pyrimidinyl 89 3-phenylpropyl ethenyl 4-isoquinolyl 90
2-phenylethenyl bond 4-sulfonylphenyl 91 3-phenylprop-2-enyl
chloro-methyl 4-imino-phenyl 92 3-bromopropyl
--CH.sub.2--N.dbd.CH-- 4-phenylethoxy-phenyl 93 4-fluoro-n-butyl
--CH.sub.2--S--CH.sub.2-- - 4-ethylphenoxy-phenyl 94
3-methoxypropyl --CH.sub.2--NH--CH.sub.2- -- 4-phenoxy-phenyl 95
2-hydroxyethyl --CH.sub.2--O--CH.sub.2-- 3-phenylpropyl-phenyl 96
tert-butyl --CH.sub.2-- 32 97 tert-butyl bond 2-chloro-phenyl 98
tert-butyl bond 4-chloro-phenyl 99 tert-butyl bond
3,4,5-trimethoxy-phenyl 100 tert-butyl bond 33 101 tert-butyl bond
34 102 tert-butyl --O--CH.sub.2--, X phenyl attaches directly to
the CH.sub.2
Example 4
[0184] Approximate IC.sub.50 Data for Selected Compounds
[0185] The IC.sub.50 of with respect to PARP inhibition was
determined for several compounds by a PARP assay using purified
recombinant human PARP from Trevigen (Gaithersburg, Md.), as
follows: The PARP enzyme assay was set up on ice in a volume of 100
microliters consisting of 10 mM Tris-HCl (pH 8.0), 1 mM MgCl.sub.2,
28 mM KCl, 28 mM NaCl, 0.1 mg/ml of herring sperm DNA (activated as
a 1 mg/ml stock for 10 minutes in a 0.15% hydrogen peroxide
solution), 3.0 micromolar [3H] nicotinamide adenine dirucleotide
(470 mci/mmole), 7 micrograms/ml PARP enzyme, and various
concentrations of the compounds to be tested. The reaction was
initiated by incubating the mixture at 25.degree. C. After 15
minutes' incubation, the reaction was terminated by adding 500
microliters of ice cold 20% (w/v) trichloroacetic acid. The
precipitate formed was transferred onto a glass fiber filter
(Packard Unifilter-GF/B) and washed three times with ethanol. After
the filter was dried, the radioactivity was determined by
scintillation counting.
[0186] Using the PARP assay described above, approximate IC.sub.50
values were obtained for the following compounds: 35
[0187] Similar IC.sub.50 values are obtained for the other
ortho-diphenol compounds of the invention.
Example 5
[0188] Neuroprotective Effect of DPQ on Focal Cerebral Ischemia in
Rats
[0189] Focal cerebral ischemia was produced by cauterization of the
right distal MCA (middle cerebral artery) with bilateral temporary
common carotid artery occlusion in male Long-Evans rats for 90
minutes. All procedures performed on the animals were approved by
the University Institutional Animal Care and Use Committee of the
University of Pennsylvania. A total of 42 rats (weights: 230-340 g)
obtained from Charles River were used in this study. The animals
fasted overnight with free access to water prior to the surgical
procedure.
[0190] Two hours prior to MCA occlusion, varying amounts (control,
n=14; 5 mg/kg, n=7; 10 mg/kg, n=7; 20 mg/kg, n=7; and 40 mg/kg,
n=7) of the compound, 3,4-dihydro-5-[4-(1-piperidinyl)-butoxy]-1
(2H)-isoquinolinone ("DPQ") were dissolved in dimethyl sulfoxide
(DMSO) using a sonicator. A volume of 1.28 ml/kg of the resulting
solution was injected intraperitoneally into fourteen rats.
[0191] The rats were then anesthetized with halothane (4% for
induction and 0.8%-1.2% for the surgical procedure) in a mixture of
70% nitrous oxide and 30% oxygen. The body temperature was
monitored by a rectal probe and maintained at 37.5.+-.0.5.degree.
C. with a heating blanket regulated by a homeothermic blanket
control unit (Harvard Apparatus Limited, Kent, U.K.). A catheter
(PE-50) was placed into the tail artery, and arterial pressure was
continuously monitored and recorded on a Grass polygraph recorder
(Model 7D, Grass Instruments, Quincy, Mass.). Samples for blood gas
analysis (arterial pH, PaO.sub.2 and PaCO.sub.2) were also taken
from the tail artery catheter and measured with a blood gas
analyzer (ABL 30, Radiometer, Copenhagen, Denmark). Arterial blood
samples were obtained 30 minutes after MCA occlusion.
[0192] The head of the animal was positioned in a stereotaxic
frame, and a right parietal incision between the right lateral
canthus and the external auditory meatus was made. Using a dental
drill constantly cooled with saline, a 3 mm burr hole was prepared
over the cortex supplied by the right MCA, 4 mm lateral to the
sagittal suture and 5 mm caudal to the coronal suture. The dura
mater and a thin inner bone layer were kept, care being taken to
position the probe over a tissue area devoid of large blood
vessels. The flow probe (tip diameter of 1 mm, fiber separation of
0.25 mm) was lowered to the bottom of the cranial burr hole using a
micromanipulator. The probe was held stationary by a probe holder
secured to the skull with dental cement. The microvascular blood
flow in the right parietal cortex was continuously monitored with a
laser Doppler flowmeter (FloLab, Moor, Devon, U.K., and Periflux
4001, Perimed, Stockholm, Sweden).
[0193] Focal cerebral ischemia was produced by cauterization of the
distal portion of the right MCA with bilateral temporary common
carotid artery (CCA) occlusion by the procedure of Chen et al., "A
Model of Focal Ischemic Stroke in the Rat: Reproducible Extensive
Cortical Infarction", Stroke 17:738-43 (1986) and/or Liu et al.,
"Polyethylene Glycol-conjugated Superoxide Dismutase and Catalase
Reduce Ischemic Brain Injury", Am. J. Physiol. 256:H589-93 (1989),
both of which are hereby incorporated by reference.
[0194] Specifically, bilateral CCA's were isolated, and loops made
from polyethylene (PE-10) catheter were carefully passed around the
CCA's for later remote occlusion. The incision made previously for
placement of the laser doppler probe was extended to allow
observation of the rostral end of the zygomatic arch at the fusion
point using a dental drill, and the dura mater overlying the MCA
was cut. The MCA distal to its crossing with the inferior cerebral
vein was lifted by a fine stainless steel hook attached to a
micromanipulator and, following bilateral CCA occlusion, the MCA
was cauterized with an electrocoagulator. The burr hole was covered
with a small piece of Gelform, and the wound was sutured to
maintain the brain temperature within the normal or near-normal
range.
[0195] After 90 minutes of occlusion, the carotid loops were
released, the tail arterial catheter was removed, and all of the
wounds were sutured. Gentamicin sulfate (10 mg/ml) was topically
applied to the wounds to prevent infection. The anesthetic was
discontinued, and the animal was returned to his cage after
awakening. Water and food were allowed ad libitum.
[0196] Two hours after MCA occlusion, the animals were given the
same doses of the PARP inhibitor as in the pre-treatment.
Twenty-four hours after MCA occlusion, the rats were sacrificed
with an intraperitoneal injection of pentobarbital sodium (150
mg/kg). The brain was carefully removed from the skull and cooled
in ice-cold artificial CSF for five minutes. The cooled brain was
then sectioned in the coronal plane at 2 mm intervals using a
rodent brain matrix (RBM-4000C, ASI Instruments, Warren, Mich.).
The brain slices were incubated in phosphate-buffered saline
containing 2% 2,3,5-triphenyltetrazolium chloride (TTC) at
37.degree. C. for ten minutes. Color photographs were taken of the
posterior surface of the stained slices and were used to determine
the damaged area at each cross-sectional level using a
computer-based image analyzer (NIH Image 1.59). To avoid artifacts
due to edema, the damaged area was calculated by subtracting the
area of the normal tissue in the hemisphere ipsilateral to the
stroke from the area of the hemisphere contralateral to the stroke,
by the method of Swanson et al., "A Semiautomated Method for
Measuring Brain Infarct Volume", J. Cereb. Blood Flow Metabol.
10:290-93 (1990), the disclosure of which is hereby incorporated by
reference. The total volume of infarction was calculated by
summation of the damaged volume of the brain slices.
[0197] The cauterization of the distal portion of the right MCA
with bilateral temporary CCA occlusion consistently produced a
well-recognized cortical infarct in the right MCA territory of each
test animal. There was an apparent uniformity in the distribution
of the damaged area as measured by TTC staining in each group, as
shown in FIG. 1.
[0198] In FIG. 1, the distribution of the cross-sectional infarct
area at representative levels along the rostrocaudal axis was
measured from the interaural line in non-treated animals and in
animals treated with 10 mg/kg of
3,4-dihydro-5-[4-(1-piperidinyl)-butoxy]-1(2H)-isoquinolinone. The
area of damage was expressed as mean.+-.standard deviation.
Significant differences between the 10 mg-treated group and the
control group were indicated (.sup.+p<0.02, .sup.++p<0.01,
.sup.++p<0.001). The 5 mg/kg and 20 mg/kg curves fell
approximately halfway between the control and the 10 mg/kg curves,
whereas the 40 mg/kg curve was close to the control. The 5, 20 and
40 mg/kg curves were omitted for clarity.
[0199] PARP inhibition led to a significant decrease in the damaged
volume in the 5 mg/kg-treated group (106.7.+-.23.2 mm.sup.3,
p<0.001), the 10 mg/kg-treated group (76.4.+-.16.8 mm.sup.3,
p<0.001), and the 20 mg/kg-treated group (110.2.+-.42.0
mm.sup.3, p<0.01), compared to the control group (165.2.+-.34.0
mm.sup.3. The data are expressed as mean.+-.standard deviation. The
significance of differences between groups was determined using an
analysis of variance (ANOVA) followed by Student's t-test for
individual comparisons.
[0200] There was no significant difference between the control and
the 40 mg/kg-treated group (135.6.+-.44.8 mm.sup.3). However, there
were significant differences between the 5 mg/kg-treated group and
the 10 mg/kg-treated group (p<0.02), and between the 10
mg/kg-treated group and the 40 mg/kg-treated group (p<0.01), as
shown in FIG. 2.
[0201] In FIG. 2, the effect of intraperitoneal administration of
3,4-dihydro-5-[4-(1-piperidinyl)-butoxy]-1(2H)-isoquinolinone on
the infarct volume was depicted graphically. The volumes of infarct
were expressed as mean.+-.standard deviation. Significant
differences between the treated groups and the control group were
indicated (.sup.+p<0.01, .sup.++p<0.001). It is not clear why
a high dose (40 mg/kg) of the PARP inhibitor, 3,4-dihydro-5-[4-
(1-piperidinyl)-butoxy]-1(2H)-isoquinol- inone, was less
neuroprotective. The U-shaped dose-response curve may suggest dual
effects of the compound.
[0202] However, overall, the in vivo administration of the
inhibitor led to a substantial reduction in infarct volume in the
focal cerebral ischemia model in the rat. This result indicated
that the activation of PARP plays an important role in the
pathogenesis of brain damage in cerebral ischemia.
[0203] The values of arterial blood gases (PaO.sub.2, PaCO.sub.2
and pH) were within the physiological range in the control and
treated groups with no significant differences in these parameters
among the five groups, as shown below in Table 2. A "steady state"
MABP was taken following completion of the surgical preparation,
just prior to occlusion; an "ischemia" MABP was taken as the
average MABP during occlusion. See Table III below:
8 TABLE III MABP (mm Hg) PaO.sub.2 PaCO.sub.2 Steady Ischemia (mm
Hg) mm Hg; pH State Control 125 .+-. 21 38.6 .+-. 4.6 7.33 .+-.
0.05 79 .+-. 14 91 .+-. 13** group (n = 4) 5 mg/kg- 126 .+-. 20
38.0 .+-. 2.8 7.36 .+-. 0.02 78 .+-. 5 91 .+-. 12** treated group
(n = 7) 10 mg/kg- 125 .+-. 16 39.3 .+-. 5.2 7.34 .+-. 0.03 80 .+-.
9 90 .+-. 14* treated group (n = 7) 20 mg/kg- 122 .+-. 14 41.3 .+-.
2.8 7.35 .+-. 0.23 79 .+-. 10 91 .+-. 12** treated group (n = 7) 40
mg/kg- 137 .+-. 17 39.5 .+-. 4.7 7.33 .+-. 0.24 78 .+-. 4 88 .+-.
12* treated group (n = 7) * = Significantly different from the
steady state value, p < 0.05. ** = Significantly different from
the steady state value, p < 0.01.
[0204] There were no significant differences in any physiological
parameter, including mean arterial blood pressure (MABP), prior to
MCA and CCA occlusion among the five groups. Although MABP was
significantly elevated following occlusion in all five groups,
there were no significant differences in MABP during the occlusion
period among the groups.
[0205] Since the blood flow values obtained from the laser doppler
were in arbitrary units, only percent changes from the baseline
(prior to occlusion) were reported. Right MCA and bilateral CCA
occlusion produced a significant decrease in relative blood flow in
the right parietal cortex to 20.8.+-.7.7% of the baseline in the
control group (n=5), 18.7.+-.7.4% in the 5 mg/kg-treated group
(n=7), 21.4.+-.7.7% in the 10 mg/kg-treated group (n=7) and
19.3.+-.11.2% in the 40 mg/kg-treated group (n=7). There were no
significant differences in the blood flow response to occlusion
among the four groups. In addition, blood flow showed no
significant changes throughout the entire occlusion period in any
group.
[0206] Following release of the carotid occlusions, a good recovery
of blood flow (sometimes hyperemia) was observed in the right MCA
territory of all animals. Reperfusion of the ischemic tissue
resulted in the formation of NO and peroxynitrite, in addition to
oxygen-derived free radicals. All of these radicals have been shown
to cause DNA strand breaks and to activate PARP.
[0207] This example provided evidence that the related compounds of
the present invention are effective in inhibiting PARP
activity.
Example 6
[0208] Assay for Neuroprotective Effects on Focal Cerebral Ischemia
in Rats
[0209] Focal cerebral ischemia experiments are performed using male
Wistar rats weighing 250-300 g, which are anesthetized with 4%
halothane. Anesthesia is maintained with 1.0-1.5% halothane until
the end of surgery. The animals are installed in a warm environment
to avoid a decrease in body temperature during surgery.
[0210] An anterior midline cervical incision is made. The right
common carotid artery (CCA) is exposed and isolated from the vagus
nerve. A silk suture is placed and tied around the CCA in proximity
to the heart. The external carotid artery (ECA) is then exposed and
ligated with a silk suture. A puncture is made in the CCA and a
small catheter (PE 10, Ulrich & Co., St-Gallen, Switzerland) is
gently advanced to the lumen of the internal carotid artery (ICA).
The pterygopalatine artery is not occluded. The catheter is tied in
place with a silk suture. Then, a 4-0 nylon suture (Braun Medical,
Crissier, Switzerland) is introduced into the catheter lumen and is
pushed until the tip blocks the anterior cerebral artery. The
length of catheter into the ICA is approximately 19 mm from the
origin of the ECA. The suture is maintained in this position by
occlusion of the catheter with heat. One cm of catheter and nylon
suture are left protruding so that the suture can be withdrawn to
allow reperfusion. The skin incision is then closed with wound
clips.
[0211] The animals are maintained in a warm environment during
recovery from anesthesia. Two hours later, the animals are
re-anesthetized, the clips are discarded, and the wound is
re-opened. The catheter is cut, and the suture is pulled out. The
catheter is then obturated again by heat, and wound clips are
placed on the wound. The animals are allowed to survive for 24
hours with free access to food and water. The rats are then
sacrificed with CO.sub.2 and decapitated.
[0212] The brains are immediately removed, frozen on dry ice and
stored at -80.degree. C. The brains are then cut in 0.02 mm-thick
sections in a cryocut at -19.degree. C., selecting one of every 20
sections for further examination. The selected sections are stained
with cresyl violet according to the Nissl procedure. Each stained
section is examined under a light microscope, and the regional
infarct area is determined according to the presence of cells with
morphological changes.
[0213] Various doses of the compounds of the invention are tested
in this model. The compounds are administered in either a single
dose or a series of multiple doses, i.p. or i.v., at different
times, both before or after the onset of ischemia. Compounds of the
invention are found to provide protection from ischemia in the
range of about 20 to 80%.
Example 7
[0214] Effects on Heart Ischemia/Reperfusion Injury in Rats
[0215] Female Sprague-Dawley rats, each weighing about 300-350 g
are anesthetized with intraperitoneal ketamine at a dose of 150
mg/kg. The rats are endotracheally intubated and ventilated with
oxygen-enriched room air using a Harvard rodent ventilator.
Polyethylene catheters inserted into the carotid artery and the
femoral vein are used for artery blood pressure monitoring and
fluid administration respectively. Arterial pCO.sub.2 is maintained
between 35 and 45 mm Hg by adjusting the respirator rate. The rat
chests are opened by median sternotomy, the pericardium is incised,
and the hearts are cradled with a latex membrane tent. Hemodynamic
data are obtained at baseline after at least a 15-minute
stabilization period following the end of the surgical operation.
The LAD (left anterior descending) coronary artery is ligated for
40 minutes, and then re-perfused for 120 minutes. After 120
minutes' reperfusion, the LAD artery is re-occluded, and a 0.1 ml
bolus of monastral blue dye is injected into the left atrium to
determine the ischemic risk region.
[0216] The hearts are then arrested with potassium chloride and cut
into five 2-3 mm thick transverse slices. Each slice is weighed and
incubated in a 1% solution of trimethyltetrazolium chloride to
visualize the infarcted myocardium located within the risk region.
Infarct size is calculated by summing the values for each left
ventricular slice and is further expressed as a fraction of the
risk region of the left ventricle.
[0217] Various doses of the compounds of the invention are tested
in this model. The compounds are given either in a single dose or a
series of multiple doses, i.p. or i.v., at different times, both
before or after the onset of ischemia. The compounds of the
invention are found to have ischemia/reperfusion injury protection
in the range of 10 to 40 percent. Therefore, they protect against
ischemia-induced degeneration of rat hippocampal neurons in
vitro.
Example 8
[0218] Retinal Ischemia Protection
[0219] A patient just diagnosed with acute retinal ischemia is
immediately administered parenterally, either by intermittent or
continuous intravenous administration, a compound of formula I,
either as a single dose or a series of divided doses of the
compound. After this initial treatment, and depending on the
patient's presenting neurological symptoms, the patient optionally
may receive the same or a different compound of the invention in
the form of another parenteral dose. It is expected by the
inventors that significant prevention of neural tissue damage would
ensue and that the patient's neurological symptoms would
considerably lessen due to the administration of the compound,
leaving fewer residual neurological effects post-stroke. In
addition, it is expected that the re-occurrence of retinal ischemia
would be prevented or reduced.
Example 9
[0220] Treatment of Retinal Ischemia
[0221] A patient has just been diagnosed with acute retinal
ischemia. Immediately, a physician or a nurse parenterally
administers a compound of formula I, either as a single dose or as
a series of divided doses. The patient also receives the same or a
different PARP inhibitor by intermittent or continuous
administration via implantation of a biocompatible, biodegradable
polymeric matrix delivery system comprising a compound of formula
I, or via a subdural pump inserted to administer the compound
directly to the infarct area of the brain. It is expected by the
inventors that the patient would awaken from the coma more quickly
than if the compound of the invention were not administered. The
treatment is also expected to reduce the severity of the patient's
residual neurological symptoms. In addition, it is expected that
re-occurrence of retinal ischemia would be reduced.
Example 10
[0222] Vascular Stroke Protection
[0223] A patient just diagnosed with acute vascular stroke is
immediately administered parenterally, either by intermittent or
continuous intravenous administration, a compound of formula I,
either as a single dose or a series of divided doses of the
compound. After this initial treatment, and depending on the
patient's presenting neurological symptoms, the patient optionally
may receive the same or a different compound of the invention in
the form of another parenteral dose. It is expected by the
inventors that significant prevention of neural tissue damage would
ensue and that the patient's neurological symptoms would
considerably lessen due to the administration of the compound,
leaving fewer residual neurological effects post-stroke. In
addition, it is expected that the re-occurrence of vascular stroke
would be prevented or reduced.
Example 11
[0224] Treatment of Vascular Stroke
[0225] A patient has just been diagnosed with acute multiple
vascular strokes and is comatose. Immediately, a physician or a
nurse parenterally administers a compound of formula I, either as a
single dose or as a series of divided doses. Due to the comatose
state of the patient, the patient also receives the same or a
different PARP inhibitor by intermittent or continuous
administration via implantation of a biocompatible, biodegradable
polymeric matrix delivery system comprising a compound of formula
I, or via a subdural pump inserted to administer the compound
directly to the infarct area of the brain. It is expected by the
inventors that the patient would awaken from the coma more quickly
than if the compound of the invention were not administered. The
treatment is also expected to reduce the severity of the patient's
residual neurological symptoms. In addition, it is expected that
re-occurrence of vascular stroke would be reduced.
Example 12
[0226] Preventing Cardiac Reperfusion Injury
[0227] A patient is diagnosed with life-threatening cardiomyopathy
and requires a heart transplant. Until a donor heart is found, the
patient is maintained on Extra Corporeal Oxygenation Monitoring
(ECMO).
[0228] A donor heart is then located, and the patient undergoes a
surgical transplant procedure, during which the patient is placed
on a heart-lung pump. The patient receives a compound of the
invention intracardiac within a specified period of time prior to
re-routing his or her circulation from the heart-lung pump to his
or her new heart, thus preventing cardiac reperfusion injury as the
new heart begins to beat independently of the external heart-lung
pump.
Example 13
[0229] Septic Shock Assay
[0230] Groups of 10 C57/BL male mice weighing 18 to 20 g were
administered a test compound, 1-carboxynaphthalene-1-carboxamide at
the doses of 60, 20, 6 and 2 mg/kg, daily, by intraperitopeal (IP)
injection for three consecutive days. Each animal was first
challenged with lipopolysaccharide (LPS, from E. Coli, LD.sub.100
of 20 mg/animal IV) plus galactosamine (20 mg/animal IV). The first
dose of test compound in a suitable vehicle was given 30 minutes
after challenge, and the second and third doses were given 24 hours
later on day 2 and day 3 respectively, with only the surviving
animals receiving the second or third dose of the test compound.
Mortality was recorded every 12 hours after challenge for the
three-day testing period. 1-Carboxy-naphthalene-1- -carboxamide
provided a protection against mortality from septic shock of about
40%. Based on these results, other compounds of the invention are
expected to provide a protection against mortality exceeding about
35%.
Example 14
[0231] Inhibition of PARP Activity
[0232] A patient has just been diagnosed with a disorder requiring
the administration of a PARP inhibitor. A physician or a nurse
parenterally administers a compound of formula I, either as a
single dose or as a series of divided doses. The patient may
receive the same or a different PARP inhibitor by intermittent or
continuous administration via implantation of a biocompatible,
biodegradable polymeric matrix delivery system comprising a
compound of formula I, or via a subdural pump inserted to
administer the compound directly to the desired treatment location.
It would be expected that the treatment would alleviate the
disorder, either in part or in its entirety and that no further
occurrences of the disorder would develop.
Example 15
[0233] A treatment such as that described in Example 14 wherein the
patient is diagnosed with a peripheral neuropathy caused by
physical injury or a disease state.
Example 16
[0234] A treatment such as that described in Example 14 wherein the
patient is diagnosed with Guillain-Barre syndrome.
Example 17
[0235] A treatment such as that described in Example 14 wherein the
patient is diagnosed with traumatic brain injury.
Example 18
[0236] A treatment such as that described in Example 14 wherein the
patient is diagnosed with physical damage to the spinal cord.
Example 19
[0237] A treatment such as that described in Example 14 wherein the
patient is diagnosed with stroke associated with brain damage.
Example 20
[0238] A treatment such as that described in Example 14 wherein the
patient is diagnosed with focal ischemia.
Example 21
[0239] A treatment such as that described in Example 14 wherein the
patient is diagnosed with global ischemia.
Example 22
[0240] A treatment such as that described in Example 14 wherein the
patient is diagnosed with reperfusion injury.
Example 23
[0241] A treatment such as that described in Example 14 wherein the
patient is diagnosed with a demyelinating disease.
Example 24
[0242] A treatment such as that described in Example 14 wherein the
patient is diagnosed with multiple sclerosis.
Example 25
[0243] A treatment such as that described in Example 14 wherein the
patient is diagnosed with a neurological disorder relating to
neurodegeneration.
Example 26
[0244] A treatment such as that described in Example 14 wherein the
patient is diagnosed with Alzheimer's Disease.
Example 27
[0245] A treatment such as that described in Example 14 wherein the
patient is diagnosed with Parkinson's Disease.
Example 28
[0246] A treatment such as that described in Example 14 wherein the
patient is diagnosed with amyotrophic lateral sclerosis.
Example 29
[0247] A treatment such as that described in Example 14 wherein the
patient is diagnosed with a cardiovascular disease.
Example 30
[0248] A treatment such as that described in Example 14 wherein the
patient is diagnosed with angina pectoris.
Example 31
[0249] A treatment such as that described in Example 14 wherein the
patient is diagnosed with myocardial infarction.
Example 32
[0250] A treatment such as that described in Example 14 wherein the
patient is diagnosed with cardiovascular tissue damage related to
PARP activation.
Example 33
[0251] In vitro Radiosensitization
[0252] The human prostate cancer cell line, PC-3s, were plated in 6
well dishes and grown at monolayer cultures in RPMI1640
supplemented with 10% FCS. The cells are maintained at 37.degree.
C. in 5% CO.sub.2 and 95% air. The cells were exposed to a dose
response (0.1 mM to 0.1 .mu.M) of 3 different PARP inhibitors of
Formula I disclosed herein prior to irradiation at one sublethal
dose level. For all treatment groups, the six well plates were
exposed at room temperature in a Seifert 250 kV/15 mA irradiator
with a 0.5 mm Cu/1 mm. Cell viability was examined by exclusion of
0.4% trypan blue. Dye exclusion was assessed visually by microscopy
and viable cell number was calculated by subtracting the number of
cells from the viable cell number and dividing by the total number
of cells. Cell proliferation rates were calculated by the amount of
.sup.3H-thymidine incorporation post-irradiation. The PARP
inhibitors show radiosensitization of the cells.
Example 34
[0253] In Vivo Radiosensitization
[0254] Before undergoing radiation therapy to treat cancer, a
patient is administered an effective amount of a compound or a
pharmaceutical composition of the present invention. The compound
or pharmaceutical composition acts as a radiosensitizer and making
the tumor more susceptible to radiation therapy.
Example 35
[0255] Measuring Altered Gene Expression in mRNA Senescent
Cells
[0256] Human fibroblast BJ cells, at Population Doubling (PDL) 94,
are plated in regular growth medium and then changed to low serum
medium to reflect physiological conditions described in Linskens,
et al., Nucleic Acids Res. 23:16:3244-3251 (1995). A medium of
DMEM/199 wupplemented with 0.5% bovine calf serum is used: The
cells are treated daily for 13 days with the PARP inhibitor of
Formula I. The control cells are treated with and without the
solvent used to administer the PARP inhibitor. The untreated old
and young control cells are tested for comparison. RNA is prepared
from the treated and control cells according to the techniques
described in PCT Publication No. 96/13610 and Northern blotting is
conducted. Probes specific for senescence-related genes are
analyzed, and treated and control cells compared. In analyzing the
results, the lowest level of gene expression is arbitrarily set at
1 to provide a basis for comparison. Three genes particularly
relevant to age-related changes in the skin are collagen,
collagenase and elastin. West, Arch. Derm. 130:87-95 (1994).
Elastin expression of the cells treated with the PARP inhibitor of
Formula I is significantly increased in comparison with the control
cells. Elastin expression is significantly higher in young cells
compared to senescent cells, and thus treatment with the PARP
inhibitor of Formula I causes elastin expression levels in
senescent cells to change to levels similar to those found in much
younger cells. Similarly, a beneficial effect is seen in
collagenase and collagen expression with treatment with the PARP
inhibitors of Formula I.
Example 36
[0257] Measuring Altered Gene Expression Protein in Senescent
Cells
[0258] Approximately 105 BJ cells, at PDL 95-100 are plated and
grown in 15 cm dishes. The growth medium is DMEM/199 supplemented
with 10% bovine calf serum. The cells are treated daily for 24
hours with the PARP inhibitors of Formula I (100 .mu.g/1 mL of
medium). The cells are washed with phosphate buffered solution
(PBS), then permeablized with 4% paraformaldehyde for 5 minutes,
then washed with PBS, and treated with 100% cold methanol for 10
minutes. The methanol is removed and the cells are washed with PBS,
and then treated with 10% serum to block nonspecific antibody
binding. About 1 mL of the appropriate commercially available
antibody solutions (1:500 dilution Vector) is added to the cells
and the mixture incubated for 1 hour. The cells are rinsed and
washed three times with PBS. A secondary antibody, goat anti-mouse
IgG (1 mL) with a biotin tag is added along with 1 mL of a solution
containing streptavidin conjugated to alkaline phosphatase and 1 mL
of NBT reagent (Vector). The cells are washed and changes in gene
expression are noted colorimetrically. Four senescence-specific
genes--collagen I, collagen III, collagenase, and interferon
gamma--in senescent cells treated with the PARP inhibitor of
Formula I are monitored and the results show a decrease in
interferon gamma expression with no observable change in the
expression levels of the other three gens, demonstrating that the
PARP inhibitors of Formula I can alter senescence-specific gene
expression.
Example 37
[0259] Extending or Increasing Proliferative Capacity and Lifespan
of Cells
[0260] To demonstrate the effectiveness of the present method for
extending the proliferative capacity and lifespan of cells, human
fibroblast cells lines (either W138 at Population Doubling (PDL) 23
or BJ cells at PDL 71) are thawed and plated on T75 flasks and
allowed to grow in normal medium (DMEM/M199 plus 10% bovine calf
serum) for about a week, at which time the cells are confluent, and
the cultures are therefor ready to be subdivided. At the time of
subdivision, the media is aspirated, and the cells rinsed with
phosphate buffer saline (PBS) and then trypsinized. The cells are
counted with a Coulter counter and plated at a density of 10.sup.5
cells per cm.sup.2 in 6-well tissue culture plates in DMEM/199
medium supplemented with 10% bovine calf serum and varying amounts
(0.10 .mu.M, and 1 mM: from a 100X stock solution in DMEM/M199
medium) of a PARP inhibitor of Formula I as disclosed herein. This
process is repeated every 7 days until the cell appear to stop
dividing. The untreated (control) cells reach senescence and stop
dividing after about 40 days in culture. Treatment of cells with 10
.mu.M 3-AB appears to have little or no effect in contrast to
treatment with 100 .mu.M 3-AB which appears lengthen the lifespan
of the cells and treatment with 1 mM 3-AB which dramatically
increases the lifespan and proliferative capacity of the cells. The
cells treated with 1 mM 3-AB will still divide after 60 days in
culture.
Example 38
[0261] Neuroprotective Effects on Chronic Constriction Injury (CCI)
in Rats
[0262] Adult male Sprague-Dawley rats, 300-350 g, are anesthetized
with intraperitoneal 50 mg/kg sodium pentobarbital. Nerve ligation
is performed by exposing one side of the rat's sciatic nerves and
dissecting a 5-7 mm-long nerve segment and closing with four loose
ligatures at a 1.0-1.5-mm, followed by implanting of an intrathecal
catheter and inserting of a gentamicin sulfate-flushed polyethylene
(PE-10) tube into the subarachnoid space through an incision at the
cisterna magna. The caudal end of the catheter is gently threaded
to the lumbar enlargement and the rostral end is secured with
dental cement to a screw embedded in the skull and the skin wound
is closed with wound clips.
[0263] Thermal hyperalgesia to radiant heat is assessed by using a
paw-withdrawal test. The rat is placed in a plastic cylinder on a
3-mm thick glass plate with a radiant heat source from a projection
bulb placed directly under the plantar surface of the rat's
hindpaw. The paw-withdrawal latency is defined as the time elapsed
from the onset of radiant heat stimulation to withdrawal of the
rat's hindpaw.
[0264] Mechanical hyperalgesia is assessed by placing the rat in a
cage with a bottom made of perforated metal sheet with many small
square holes. Duration of paw-withdrawal is recorded after pricking
the mid-plantar surface of the rat's hindpaw with the tip of a
safety pin inserted through the cage bottom.
[0265] Mechano-allodynia is assessed by placing a rat in a cage
similar to the previous test, and applying von Frey filaments in
ascending order of bending force ranging from 0.07 to 76 g to the
mid-plantar surface of the rat's hindpaw. A von Frey filament is
applied perpendicular to the skin and depressed slowly until it
bends. A threshold force of response is defined as the first
filament in the series to evoke at least one clear paw-withdrawal
out of five applications.
[0266] Dark neurons are observed bilaterally within the spinal cord
dorsal horn, particularly in laminae I-II, of rats 8 days after
unilateral sciatic nerve ligation as compared with sham operated
rats. Various doses of differing compounds of Formula I tested in
this model and show that the Formula I compounds reduce both
incidence of dark neurons and neuropathic pain behavior in CCI
rats.
[0267] The invention being thus described, it will be obvious that
the same may be varied in many ways. Such variations are not to be
regarded as a departure from the spirit and scope of the invention,
and all such modifications are intended to be included within the
scope of the following claims.
* * * * *