Note: Descriptions are shown in the official language in which they were submitted.
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METHODS FIND COMPOSITIONS OF NOVEL
r1'RIAIINE COMPOUNDS
FIELD OF TILE INVIV,N~I'lON
The present invention relates to triazine compounds. More particularly,
the invention relates to methods and compositions for making and using
triazine
compounds.
F3ACKGROUND OF TIIF1NVFNTION
Synthesis of novel compounds leads to new possibilities for
discovery of novel therapeutic interventions. By using structure and activity
relationship investigations, compounds can be tailored so that the compounds
have at least one activity that can be predicted from its structure. Using
high-
throughput assays allows for the rapid determination of the activity of the
newly
synthesized compounds.
Novel compounds for new therapeutic interventions are needed for
many areas of medicine and disease treatment. For example, chronic and acute
inflammatory conditions form the basis for diseases affecting all organ
systems
including, but not limited to, asthma, acute inflammatory diseases, vascular
inflammatory disease, chronic inflammation, atherosclerosis, angiopathy,
myocarditis, nephritis, Crohn's disease, arthritis, type I and II diabetes and
associated vascular pathologies. The incidence of these inflammatory
conditions
is on the rise in the population as a whole, with diabetes alone affecting 16
million people.
While inflammation in and of itself is a normal immune response,
chronic inflammation leads to complications and ongoing system damage due to
the interactions of unknown cellular factors. In particular, chronic
inflammation
can cause endothelial damage resulting in vascular complications. Coronary
artery, cerbrovascular and peripheral vascular disease resulting from
atherosclerotic and thromboembolic macroangiopathy are the primary causes of
mortality in chronic inflammatory diseases.
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Many humans and animals have limited lifespans and lifestyles
because of conditions relating to lifestyle choices, such as diet and
exercise, or
because of genetic predispositions to develop a disease. For example, vascular
smooth muscle cell proliferation is a common consequence of endothelial injury
and is believed to be an early pathogenetic event in the formation of
atherosclerotic plaques or complications related to vascular injury or as a
result
surgical interventions. Abnormal vascular smooth muscle cell (SMC)
proliferation is thought to contribute to the pathogenesis of vascular
occlusive
lesions, including arteriosclerosis, atherosclerosis, restenosis, and graft
atherosclerosis after organ transplantation.
Percutaneous coronary artery intervention (PTCA) procedures are
the most common in-patient hospital procedure in the United States. According
to the American Heart Association, about one-third of the patients that
undergo
balloon angioplasty have restenosis of the widened segment of the vessel
within
approximately 6 months. It may be necessary to perform another angioplasty or
coronary artery bypass surgery on restenosed arteries. A key feature of
restenosis
is an injury response that results in activation of an inflammatory cascade
and
remodeling of the cells both inside and outside the carotid artery wall. This
includes excessive growth of connective tissue and smooth muscle into the
lumen
of the artery known as neointimal hyperplasia. Currently there are no
effective
pharmacological treatments available that control the pathogenesis of vascular
occlusive lesions, such as, but not limited to, arteriosclerosis,
atherosclerosis,
restenosis, and graft atherosclerosis after organ transplantation.
Identification of
effective therapeutics with minimal side effects will restore quality of life
without
requiring additional surgical procedures such as coronary artery bypass
surgery.
Control or modulation of factors produced by the body in response
to injury, surgery, metabolic factors or loss of control of in feedback
mechanisms,
leading to too much or too little of a factor has long been the goal of
administering pharmacological agents. One disease that rapidly growing in the
industrialized countries is the occurrence of diabetes and all of its
attendant
sequellae. One of the factors important in the damage associated with diabetes
is
the presence of glycated proteins.
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Glycated proteins and advanced glycation end products (AGE)
contribute to cellular damage, particularly, diabetic tissue injury, by at
least by
two major mechanisms; modulation of cellular functions through interactions
with specific cell surface receptors, and alteration of the extracellular
matrix
leading to the formation of protein cross-links. Studies suggest that glycated
protein and AGE interactions with cells may promote inflammatory processes
and oxidative cellular injury. AGE increases lipoprotein oxidisability and
atherogenicity. Its binding to matrix proteins induces synthesis of cytokines
and
activates cellular messangers. Diseases where glycated protein and AGE
accumulation is a suspected etiological factor include vascular complications
of
diabetes, microangiopathies, renal insufficiency and Alzheimer's disease.
The exact mechanisms by which high plasma glucose, as seen in
diabetes, causes microvascular damage are not completely understood. One
potential mechanism by which hyperglycemia can be linked to microangiopathies
is through the process of non-enzymatic glycation of critical proteins. Non-
enzymatic glycation, i.e. the linking of proteins with glucose, leads to the
formation of glycated proteins. The first step in this glycation pathway
involves
the non-enzymatic condensation of glucose with free amino groups in the
protein,
primarily the epsilon-amino groups of lysine residues, forming the Amadori
adducts. These early glycation products can undergo further reactions such as
rearrangements, dehydration and condensations to form irreversible advanced
glycation end products (AGE). These are a highly reactive group of molecules
whose interaction with specific receptors on the cell-surface which are
thought to
lead to pathogenic outcomes.
~5 Other major area of disease of where treatments are needed and
for which adequate and effective therapies do not exist are cellular
proliferative
disorders, or disorders caused by unwanted or unintended cellular growth.. As
mentioned, smooth muscle cell (SMC) hyperplasia is a major event in the
development of atherosclerosis and is also responsible for the significant
number
of failure rates following vascular procedures such as angioplasty, stmt
implantation and coronary artery bypass surgery. In the normal vessel, SMC are
quiescent, but they proliferate when damage to the endothelium occurs.
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Naturally occurring growth modulators, many of which are derived from the
endothelium, tightly control SMC proliferation in vivo. When the control
becomes unregulated, a pathological state is induced in the subject.
Another major area of unwanted cellular growth, that is unchecked
by the body's regulatory systems, is cancer or ontological conditions. Many
therapies have been used and are being used in an effort to restore health or
at
least stop the unwanted cell growth. Many times, therapeutic agents can have
an
effect individually, but often, therapeutic regimes require combinations of
different pharmacological agents with treatments such as surgery or radiation.
There is a present need for treatments of chronic or acute diseases,
such as atherosclerosis, unwanted cellular growth or cellular proliferation,
diabetes, inflammatory conditions and vascular occlusive pathologic
conditions,
because occurrence is frequent, the currently available treatments are costly
and
the conditions are refractory to many pharmacological therapies. The
mechanisms involved in the control or prevention of such diseases are not
clear
and there exists a need for preventive and therapeutic treatments of these and
other diseases Thus, what is presently needed are novel compounds that find
utility in methods and compositions for treatment and prevention of chronic
and
acute diseases.
SUMMARY OF THE INVENTION
The present invention is directed to methods and compositions comprising
novel compounds, primarily based on a substituted triazine core. Disclosed
herein are methods for making novel compounds, the compounds, compositions
comprising the compounds, and methods and compositions for using the
compounds. The compounds and compositions comprising the compounds have
utility in treatment of a variety of diseases.
Compositions in accordance with the present invention comprise
triazine compounds, analogs, derivatives, and mixtures thereof. Such triazine
compounds comprise the following structure, where NA, NB and Nc are typically
used to represent pendant substituted amino groups attached to 1, 3, 5-
triazine at
the 2, 4 and 6 positions:
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N NA
N' /N
~%
NB
An example of such triazine compounds includes compounds
having the following structure.
R~
R2-N
-N Rs
N ~~-N-R4
>=N
R6-N
R5
In this example, each pendent amino (NRR') group can represent
simply an NHZ group or a secondary or tertiary amino group, including a cyclic
secondary amide, and a range of other substituents as described herein.
Compositions in accordance with the present invention also comprise analogs of
the tris(amino) compounds, that include intermediate compounds in the
synthesis
of the tris(amino) triazine compounds indicated above, for example diamino
chlorotriazine compounds, or amino diclorotriazine compounds shown below,
where NA and NB are pendant substituted amino groups as described above.
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NB ~ CI CI N\ CI
N ~N N /N
NA NA
Compositions in accordance with the present invention also
comprise analogs of the tris(amino) triazine compounds indicated above,
including compounds that are isolated as byproducts in the synthesis of the
tris(amino) triazine compounds, such as bis(amino)alkoxy triazine compounds as
shown below, where E = O or S and the like.
NB ~ ER
N /N
NA
The present invention also comprises compositions used in making the
novel compounds and methods of making the novel compounds disclosed herein.
The present invention is directed to methods and compositions comprising
compounds that have utility in treatment of pathological conditions. One
aspect
of the present invention comprises compounds and compositions comprising such
compounds in methods for treating diseases related to unwanted cellular
proliferation. Many vascular diseases, such as cardiovascular diseases, organ
transplant sequellae, vascular occlusive conditions including, but not limited
to,
neointimal hyperplasia, restenosis, transplant vasculopathy, cardiac allograft
vasculopathy, atherosclerosis, and arteriosclerosis. are caused by or have
collateral damage due to unwanted cellular proliferation, such as smooth
muscle
cell (SMC) hyperplasia. At least one activity of one or more of these
compounds
is that the compound has the activity of effecting the synthesis of
proteoglycans
including induction and synthesis of proteoglycans and active fragments of
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proteoglycans. Methods comprise administration of compositions comprising
compounds that have at least the activity of effecting cellular proliferation
and
effecting proteoglycan synthesis and activity.
The present invention also comprises methods and compositions
comprising compounds described herein that have an activity associated with
modulation of glycosidase enzymes and thus, effecting the substrates for such
enzymes. Glycosidase enzymes and their activity with their substrates, such as
proteoglycans or glycated proteins, are aspects of a variety of diseases such
as
vascular conditions, proteoglycan-associated diseases, kidney disease,
autoimmune disease and inflammatory diseases. Compounds described herein
that have an activity that effects the concentrations of substrates of
glycosidase
enzymes are used in methods of treatment of such vascular, inflammatory,
metastatic and systemic diseases.
An embodiment of the present invention comprises methods and
compositions comprising compounds of the present invention for the treatment
and prevention of conditions or diseases that have as an aspect of the disease
or
condition, inflammation. An aspect of the present invention is directed to
methods and compositions comprising compounds that are effective in inhibiting
inflammation, particularly inflammation associated with the accumulation or
presence of glycated proteins or AGE. Methods of treatment comprise
administration of compositions comprising having compounds having at least the
activity of modulating inflammatory reactions that are components of
biological
conditions including, but not limited to, vascular complications of type I and
type
II diabetic-induced vasculopathies, other vasculopathies, microangiopathies,
renal
insufficiency, Alzheimer's syndrome, and inflammation-induced diseases such as
atherosclerosis. An aspect of the present invention comprises methods and
compositions for the treatment of diseases, preconditions or pathologies
associated with inflammatory cytokines and other inflammation related
molecules.
Another embodiment of the present invention comprises methods and
compositions comprising compounds that~have at least the activity of causing
cellular death or a cessation of cellular activity, referred to herein as
cytotoxic
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activity. This activity can be used in methods for i~ vitro or irc vivo
cytotoxicity.
For example, compounds having this activity can be selectively delivered to an
area within a living organism to selectively kill cells in that area. Such
methods
are using in treating hyperproliferative cells, such as cancers, or other
unwanted
cellular growth or cellular activities. One aspect of the invention provides
compositions comprising compounds that nonselectively kill cells. Another
aspect of the invention provides compounds that selectively kill cells, for
example, cells that have a particular cellular marker or other identifying
characteristic such as metabolic rate or uptake of a particular compound.
The present invention also comprises pharmaceutical compositions
comprising the compounds disclosed herein. Routes of administration and
dosages of effective amounts of the compounds and pharmaceutical compositions
are also disclosed. For example, the compounds of the present invention can be
administered in combination with other pharmaceutical agents in a variety of
protocols for effective treatment of disease.
In another aspect, the present invention relates to drug delivering or
eluting medical devices that contain or are coated with at least one compound
disclosed herein. The medical device suitable for use with the compounds of
the present invention include, but are not limited to, stems and other medical
devices that can provide a substrate for delivery of at least one compound.
Other aspects of the present invention comprise compositions and
methods for microarray devices. Such microarray devices and methods
comprise a variety of microarrays that may be used, for example, to study and
monitor gene expression in response to treatment with the compounds of the
present invention. The microarrays may comprise nucleic acid sequences,
carbohydrates or proteins that are determinative for specific cells, tissues,
species, disease states, prognoses, disease progression, or any other
combination of molecules that can be used to determine an effect of one or
more of the compounds of the present invention. Other embodiments of the
present invention comprise methods using databases and computer
applications.
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BRIEF DESCRIPTION OF THE FIGURES
Figure I . 'H NMR of N-(3-Chloro-4-methoxy-phenyl)-N'-
cyclohexylmethyl-N"-methyl-N"-( I -methyl-piperidin-4-yl)-
[ 1,3,5]triazine-2,4,6-triamine.
Figure 2. 'H NMR ofN-Cycloheptyl-N'-(1-ethyl-pyrrolidin-2-ylmethyl)-
N"-(3-fluoro-4-methoxy-phenyl)-[ I ,3,5]triazine-2,4,6-triamine.
Figure 3. 'H NMR of N-(3-Chloro-4-methoxy-phenyl)-N'-methyl-N'-(1-
methyl-piperidin-4-yl)-N"-( I -propyl-butyl)-[ 1,3,5]triazine-
2,4,6-triamine.
Figure 4. 'H NMR ofN-(I-Aza-bicyclo[2.2.2]oct-3-yl)-N'-(3-chloro-4-
methoxy-phenyl)-N"-) 1-ethyl-pyrrolidin-2-ylmethyl)-
[1,3,5]triazine-2,4,6-triamine.
Figure 5. 'H NMR of N2-(3-chloro-4-methoxy-phenyl)-N4-cycloheptyl-
N6-methyl-N6-piperidin-4-yl-1,3,5-triazine-2,4,6-triamine.
Figure 6. 'H NMR of N-Cycloheptyl-N'-ethyl-N"-(3-fluoro-4-methoxy-
phenyl)-[ 1,3,5]triazine-2,4-diamine.
Figure 7. 'H NMR ofN-Cycloheptyl-N'-(3-fluoro-4-methoxy-phenyl)-6-
pyrrolidin-1-yl-[1,3,5]triazine-2,4-diamine.
Figure 8. 'H NMR ofN-Cyclohexylmethyl-N'-(1-ethyl-pyrrolidin-2-
ylmethyl)-N"-(3-fluoro-4-methoxy-phenyl)-[ 1,3,5]triazine-
2,4,6-triamine.
Figure 9. 'H NMR of 6-Chloro-N-cycloheheptyl-N'-(3-fluoro-4-
methoxy-phenyl)-[ I ,3,5]triazine-2,4-diamine.
Figure 10. 'H NMR of (3-Chloro-4-methoxy-phenyl)-(4,6-dichloro-
[ 1,3,5]triazin-2-yl)-amine.
Figure 11. 'H NMR ofN-(3-Chloro-4-methoxy-phenyl)-N'-isopropyl-N"-
methyl-N"-( 1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-
triamine.
Figure 12. 'H NMR of N2-(3-chloro-4-methoxy-phenyl) N4-isopropyl-
N6-methyl-N6-piperidin-4-yl-1,3,5-triazine-2,4,6-triamine.
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Figure 13. 'H NMR of 5-{4-(3-Chloro-4-methoxy-phenylamino)-6-
[methyl-( 1-methyl-piperidin-4-yl)-amino]-[ 1,3,5]triazin-2-
ylamino}-pentan-I-ol.
Figure 14. 'H NMR of 5-[4-(3-chloro-4-methoxy-phenylamino)-6-
(methyl-piperidin-4-yl-amino)-1,3,5-triazin-2-ylamino]-pentan-
1-ol.
Figure I5. 'H NMR of 6-Chloro-N,N"-bis-(3-chloro-4-methoxy-phenyl)-
[ 1,3,5]triazine-2,4-diamine.
Figure 16. 'HNMRofN,N'-Bis-(3-chloro-4-methoxy-phenyl)-N"-methyl-
N"-(4-methyl-cyclohexyl)-[1,3,5]triazine-2,4,6-triamine.
Figure 17. 'H NMR ofN,N'-Bis-(3-chloro-4-methoxy-phenyl)-N"-
cycloheptyl-[ I ,3,5]triazine-2,4,6-triamine.
Figure 18. 'H NMR ofN-Butyl-N'-(3-chloro-4-methoxy-phenyl)-N"-(1-
methyl-piperidin-4-yl)-N-propyl-[ 1,3,5]triazine-2,4,6-triamine.
I 5 Figure 19. 'H NMR of N2-Butyl-N4-(3-chloro-4-methoxy-phenyl) N6-
methyl-N6-piperidin-4-yl-N2-propyl-1,3,5-triazine-2,4,6-
triamine.
Figure 20. 'H NMR of 6-Cyclohexylmethoxy-N,N'-bis-(3-fluoro-4-
methoxy-phenyl)-1,3,5-triazine-2,4-diamine.
Figure 21. 'H NMR of (4-Chloro-6-cyclohexylmethoxy-[1,3,5]triazin-2
yl)-(3-fluoro-4-methoxy-phenyl)-amine.
Figure 22. 'H NMR ofN,N'-Bis-(3-chloro-4-methoxy-phenyl)-6-
cyclohexylmethoxy-1,3,5-triazine-2,4-diamine.
Figure 23. 'H NMR of (4-Chloro-6-cyclohexylmethoxy-[1,3,5]triazin-2-
yl)-(3-chloro-4-methoxy-phenyl)-amine.
Figure 24. 'H NMR of 6-Cyclohexylmethoxy-N-(I-ethyl-pyrrolidin-2-
ylmethyl)-N'-(3-fluoro-4-methoxy-phenyl)-[1,3,5]triazine-2,4-
diamine.
Figure 25. 'H NMR of N-(3-Chloro-4-methoxy-phenyl)-6-
cyclohexylmethoxy-N'-methyl-N'-( 1-methyl-piperidin-4-yl)-
[1,3,5]triazine-2,4-diamine.
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Figure 26. 'H NMR of N-Azepan-1-yl-N'-(3-chloro-4-methoxy-phenyl)-
N"-( 1-methyl-piperidin-4-yl)-[ I ,3,5]triazine-2,4,6-triamine.
Figure 27. 'H NMR of N4-(3-chloro-4-methoxy-phenyl)-N6-methyl-N2-
perhydro-azepin-I -yl-N6-piperidin-4-yl-I ,3,5-triazine-2,4,6-
triamine.
Figure 28. 'H NMR of N-Azepan-1-yl-6-chloro-N'-(3-chloro-4-methoxy-
phenyl)-[ I ,3,SJtriazine-2,4-diamine.
Figure 29. 'H NMR of N"-(3-chloro-4-methoxy-phenyl)-N,N'-bis-
perhydro-azepin- I -yl-1, 3, S-tri azine-2,4, 6-triamine.
Figure 30. 'H NMR of N-(3-Bromo-4-methoxy-phenyl)-N'-cycloheptyl-
N"-methyl-N"-( I -methyl-piperidin-4-yl)-[ 1,3,SJtriazine-2,4,6-
triamine.
Figure 31. 'H NMR ofN-(1-benzyl-piperidin-4-yl) N'-(3-chloro-4-
methoxy-phenyl)-N"-cycloheptyl-[ I ,3,SJ-2,4,6-triamine.
Figure 32. 'H NM.R of 2-chloro-4- f 4-cycloheptylamino-6-[methyl-(1-
methyl-piperidin-4-yl-aminoJ-1,3,5-triazin-2-ylamino)-phenol.
Figure 33. 'H NMR ofN2-cycloheptyl-N4-((S)-I-ethyl-pyrrolidin-2-
ylmethyl)-N6-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-
triamine.
Figure 34. 'H NMR ofN2-cycloheptyl-N4-((R)-1-ethyl-pyrrolidin-2-
ylmethyl)-N6-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-
triamine.
Figure 35. 'H NMR of N2-cyclohexylmethyl-N4-((S)-1-ethyl-pyrrolidin-2-
ylmethyl)-N6-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-
triamine.
Figure 36. 'H NMR of N2-cyclohexylmethyl-N4-((R)-1-ethyl-pyrrolidin-
2-ylmethyl)-N6-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-
2,4,6-triamine.
Figure 37. 'H NMR of ( f 4-cycloheptylamino-6-[((S)-1-ethyl-pyrrolidin-2-
ylmethyl)-amino]-1,3,5-triazin-2-ylJ-phenyl-amino)-
acetonitrile.
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Figure 38. 'H NMR of (~4-cycloheptylamino-6-[((R)-I-ethyl-pyrrolidin-2-
ylmethyl)-amino]-1,3,5-triazin-2-yl )-phenyl-amino)-
acetonitrile.
Figure 39. 'H NMR ofN2-[(1-ethyl-2-pyrrolidinyl]-N4-(3-fluoro-4-
methoxyphenyl)-6-[(S)-2-(methoxymethyl)-I-pyrrolidinyl]-
1,3,5-triazine-2,4-diamine.
Figure 40. 'H NMR of 6-Chloro-N-(3-chloro-4-methoxy-phenyl)-N'-
cycloheptyl-[ I ,3,5]triazine-2,4-diamine.
Figure 41. 'H NMR of N-(3-Chloro-4-methoxy-phenyl)-N'-cycloheptyl-
N"-methyl-N"-( I -methyl-piperidin-4-yl)-[
1,3,5]triazine-2,4,6-
triamine.
Figure 42. 'H NMR of 4-(3-Chloro-4-methoxy-phenylamino)-6-
cycloheptylamino-1,3,5-triazin-2-ol.
Figure 43. 'H NMR ofN2-(3-chloro-4-diethylamino-phenyl)-N4-
I 5 cycloheptyl-N6-( 1-ethyl-pyrrolidin-2-ylmethyl)-1,3,5-triazine-
2,4,6-triamine.
Figure 44. ' H NMR of N2-cycloheptyl-N4-(2-dimethylamino-ethyl)-N6-
(3-fluoro-4-methoxy-phenyl)-1,3,5-triazine-2,4,6-triamine.
Figure 45. 'H NMR of ( f 4-cycloheptylamino-6-[1-ethyl-pyrrolidin-2-
ylmethyl)-amino]- I ,3,5-triazin-2-yl )-phenyl-amino)-
acetonitrile.
Figure 46. 'H NMR ofN,N'-di-n-propyl-N"-(3-fluoro-4-methoxy-
phenyl)-1,3,5-triazine-2,4,6-triamine.
Figure 47. 'H NMR ofN,N'-dicyclopropyl-N"-(3-fluoro-4-methoxy-
phenyl)-1,3,5-triazine-2,4,6-triamine.
Figure 48. 'H NMR ofN2-Cycloheptyl-N4-(3-fluoro-4-methoxy-phenyl)-
N6-methyl-N6-(I-methyl-piperidin-4-yl)-I,3,5-triazine-2,4,6-
triamine.
Figure 49. ' H NMR of N2-Cycloheptyl-N4-(3-fluoro-4-methoxy-phenyl)-
N6-methyl-N6-piperidin-4-yl-1,3,5-triazine-2,4,6-triamine.
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Figure 50. 'H NMR of N2-cycloheptyl-N4-(3-fluoro-4-methoxyphenyl)-
N6-methyl-N6-( I -methyl-piperidin-4-yl)-I ,3,5-triazine-2,4,6-
triamine, hydrogen chloride salt.
Figure 51. 'H NMR ofN2-(3-chloro-4-diethylamino-phenyl)-N4-
cycloheptyl-N6-( 1-ethyl-pyrrolidin-2-ylmethyl)-1,3,5-triazine
2,4,6-triamineS42-63 hydrogen chloride salt.
Figure 52. 'H NMR of N2-cycloheptyl-N4-(I-ethyl-pyrrolidin-2-
yhnethyl)-N6-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-
triamine hydrogen chloride salt.
Figure 53. Chart showing the effects of compounds in an assay where
glycated human serum albumin (G-HSA) induces IL-6
production:
Figure 54. Chart showing the effects of compounds in an
antiproliferative assay.
DETAILED DESCRIPTION OF THE INVENTION
It is to be understood that this invention is not limited to the
particular methodology, protocols, cell lines, constructs, and reagents
described
herein and as such may vary. 1t is also to be understood that the terminology
used herein is for the purpose of describing particular embodiments only, and
is
not intended to limit the scope of the present invention which will be limited
only
by the appended claims.
All publications and patents mentioned herein are incorporated
herein by reference for the purpose of describing and disclosing, for example,
the
constructs and methodologies that are described in the publications, which
might
be used in connection with the presently described invention. The publications
discussed above and throughout the text are provided solely for their
disclosure
prior to the filing date of the present application. Nothing herein is to be
construed as an admission that the inventors are not entitled to antedate such
disclosure by virtue of prior invention.
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I. DESCRIPTION OF COMPOUNDS
In one aspect, the present invention encompasses novel organic
compounds that are generally described as Nz, 1Vø, IV6-tris(amino)-1,3,5-
triazines
which are represented by the names in Table I and the structural formulas in
the
remaining Tables 2 and following. Representative compounds of this invention
can be described by the general structural formula below, where NA, NB and N~
are pendant substituted amino groups attached to 1,3,5-triazines at the 2, 4
and 6
positions.
NC
l0
Thus, the typical compound encompassed by the present invention
includes triazine compounds comprising the following structure:
R~
R2-N
-N
N ~>--N-R4
>=N
R6-N
R5
In this typical embodiment, each pendant NR1R2, NR3R4, and
NRSR6 amino group can represent a primary, secondary, or tertiary amine when
bonded to the triazine core, including a cyclic secondary amide substitutent
(for
example a pyrrolidin-N y1 group), and a range of other substituents as
described
herein. Compositions in accordance with the present invention also comprise
analogs of the tris(amino) compounds, for example, compounds that are prepared
as intermediate compounds in the synthesis of the tris(amino) triazine
compounds
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indicated above, or compounds that represent a partially substituted trizaine
core.
Many of the syntheses of triazine compounds of this invention typically use
cyanuric chloride C3N3CI3 as a starting compound, therefore intermediate
species
such as bis(amino) chlorotriazine compounds, or amino diclorotriazine
compounds shown below, where NA and NB are pendant substituted amino
groups as described above, are also encompassed by this invention.
NB ~ CI CI ~ CI
N ~N N /N
NA NA
Compositions in accordance with the present invention also
comprise analogs of the tris(amino) triazine compounds indicated above,
including compounds that are isolated as byproducts in the synthesis of the
tris(amino) triazine compounds, a general formula of which is shown below,
where E = O or S. An example of such a compound is a bis(amino)alkoxy
triazine compound.
N~ ~ ER
N ~N
NA
In general terms, the compounds and compositions in accordance
with the present invention comprise analogs of the tris(amino) triazine
compounds of the following general structure:
CA 02461074 2004-03-19
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A
or an ene, a dime, a triene, or an yne derivative thereof; a saturated
derivative
thereof; a stereoisomer thereof; or a salt thereof;
wherein:
R' is in each occurance independently selected from -H; alkyl,
cycloalkyl, alkenyl, cycloalkenyl, cycloalkadienyl, alkynyl, aralkyl,
aralkenyl,
aralkynyl, heteroalkyl, alkoxy, alkylthio, alkylamino, or dialkylamino, each
of
which having up to 12 carbon atoms and including linear or branched
derivatives
thereof, cyclic derivatives thereof, substituted derivatives thereof,
heteroatom
derivatives thereof, or heterocyclic derivatives thereof; aryl; heteroaryl;
aryloxy;
arylthio; halogen; or amino;
G is selected from NR' or O;
E is selected from CH or N;
z is an integer from 0 to 3;
I S X' is selected from R', NR'3+, CN, N02, C02R', C(O)NR'2,
CH=CR'2, C=CR', C(O)RD, SO2R', S02OR', or NC(O)R', or X' and X2 together
is a fused aryl, pyridine, dioxane, pyrrole, pyrrolidine, furan, or thiophene
ring;
with the proviso that the R' moiety of the C(O)R' substituent in the X'
position
excludes amino or dialkylamino when X~ is C(O)R';
Xz is selected from R'; CXxH3_X, wherein X is a halogen and x is
an integer from 0 to 3; ORS; SR'; NR'2; CN; C(O)OR'; NC(O)R'; 4-morpholinyl;
4-methyl-I-piperizinyl; ORa, wherein R2 is selected from CH20CH3,
CHZOCH20CH3, CH~,OCHaCHZOCH3, CH2SCH3, or C(O)R'; SR3, wherein R3 is
selected from CHZOCH3, CHZOCHaCH20CH3, CH20CHZCH(CH3)2,
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CHzNHC(O)CH3, or SR'; OM or SM, wherein M is selected from Li, Na, K, Mg,
or Ca;
AY' is halogen, or A is selected from NR' or O, and
Y' is selected from R'; CR43; NR42; OR4; or SR4~ Z2 ~m
> >
~ Zn
~~z2 ,
Z\ I m~Zm Z~ %Z
Z' , or Zm , wherein n is an integer from 0 to 8, m is an integer
from I to 8, Z' is independently selected from CR' or N, Za is independently
selected from CR'2, NR', O, or S, with the proviso that two O or S atoms are
not
located adjacent to each other, and the proviso that no more than two Z2
moieties
are NR';
R4 is in each occurrence independently selected from linear or
branched alkyl, cycloalkyl, cycloalkenyl, cycloalkadienyl, alkenyl, alkynyl,
aralkyl, aralkenyl, aralkynyl, heteroalkyl, alkoxy, alkylthio, alkylamino, or
dialkylamino, each of which having up to 10 carbon atoms, -H, aryl,
heteroaryl,
aryloxy, arylthio, halogen, amino, NR'2-substituted derivatives thereof, OR'-
I S substituted derivatives thereof, SR'-substituted derivatives thereof, or
halogen-
substituted derivatives thereof; and
DYE is halogen, or D is selected from NR' or O wherein Rl is
defined as above, and
1 ~fIR4
Z2' ~~2 ~2'~~~2
~~2 ~2 ~2
Ya is selected from R', ~Z~ ~ ~Z2~ , or
~~ HR4
Z2 Z~Z2
Z2 Z2 , wherein Z' is independently selected from N or CR4 and Za is
independently selected as defined above, with the proviso that two O or S
atoms
17
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are not located adjacent to each other, and with the proviso that no more than
two
Zz moieties are NR~. The compounds of the present invention according to this
general description do not include those that encompass the unique combination
of substituents that would provide the following compounds:
N Cycloheptyl-N'-methyl-N'-(lanethyl-piperidin-4-yl) N"-naphthalen-2-
yl-[1,3,5]triazine-2,4,6-triamine;
N Cycloheptyl-N'-(3-fluoro-4anethoxy-phenyl)-N"-methyl-N"-(1-methyl-
piperidin-4-yl)-[ 1,3,5]triazine-2,4,6-triamine;
[4-(4-Benzyl-piperazin-I-yl)-6-morpholin-4-yl-[1,3,5]triazin-2-yl]-(4-
methoxy-phenyl)-amine;
N Cycloheptyl-6-morpholin-4-yl-N'-naphthalen-2-yl-[1,3,5]triazine-2,4-
diamine;
N Cycloheptyl-N'-(3-fluoro-4-methoxy-phenyl)-6-morpholin-4-yl-
[1,3,5]triazine-2,4-diamine;
N Cycloheptyl-6-morpholin-4-yl-N'-phenyl-[1,3,5]triazine-2,4-diamine;
N Cycloheptyl-N'-(4-methoxy-phenyl)-6-morpholin-4-yl-[1,3,5]triazine-
2,4-diamine;
N Benzyl-N'-cycloheptyl-N"-(4-methoxy-phenyl)-N methyl-
[1,3,5]triazine-2,4,6-triamine;
N (2-[1,3]Dioxolan-2-yl-ethyl)-N'-methyl-N'-(1-methyl-piperidin-4-yl)-
N"-naphthalen-2-yl-[1,3,5]triazine-2,4,6-triamine;or
N Cyclopropyl-N'-methyl-N'-(I-methyl-piperidin-4-yl) N"-naphthalen-2-
yl-[1,3,5]triazine-2,4,6-triamine.
Because this invention encompass compounds that represent
saturated derivatives of the above general structure, and compounds that
include
various states of unsaturation (for example, -ene, -dime, -triene, and -yne
derivatives of the above compounds), then the aryl or pyridyl ring shown in
the
general formula above can be partially or completed hydrogenated in this
invention. As a result, the CSE ring in the above structure can represent a
cylcohexyl or piperidynl ring that is X' and X2 substituted. In general terms,
X'
usually, but not always, represent an electron withdrawing group such as
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halide or nitro, while Xa usually, but not always, represents an electron
donating group such as alkoxide or amino.
As indicated in the general structure above, AY' and DYz
typically represent an NR' moiety (where R' is defined above), in which case
these substituents constitute a portion of amino or substituted amino group
and
therefore, the compound itself constitutes a triazine. In this case, Y' and Y2
may
be selected from a wide range of substituents, including, but not limited to,
(CH2)n
N
cycloallcyl with up to 10 carbon atoms; wherein n is from 1 or 2;
~~CHR'
s
w
I X1 NJ NR~
\\X2 ; R' ; ;linear or branched alkyl with up to 10 carbon
atoms; CH2R'; (CHR')xNR'Z wherein x is 1-6; CH2R'; (CHR')XOR' wherein x
N
I
N
. H2C~ (CH~)X herein x is rom 3 to 5' ~~ - CH CF '
is from 1 to 6, w f , , 2 3,
O
CHR' Z' wherein x is from 1 to 6 and Z' is selected from NR' ,
( )X a
I O
N ~N ~N~N
H2C~ (CH2)y wherein y is from 3 to 5, , or U . In these
examples, R' is independently selected as defined above. These are merely
I S representative examples of the definitions of the Y' and Ya substitutents,
and
are not intended to be exclusive.
It is also noted that AY' together, and DY2 together, can also
represent a wide range of chemical moieties bonded to the triazine core such
as
N I
N
ar ~~ or HaC/ (CH2)x the
halide or second y amino groups such as . In
latter cases, the amino substituent groups are termed secondary amino groups,
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however upon bonding to the triazine core, the amino nitrogens become
tertiary amine moieties. Examaples of AYI together and DYa together include,
N N ~ N
but are not limited to: halide' ~W I ~ ~ H2C/ (CH~)X w
herein x is
~~CH2)xOR1.
~N ~ N N Z2
from 3 to 5; ~ wherein x is from 0 to 6;
~N
wherein Z is selected from R , ,
O
> > ~~N~ O
C(O)R , C(O)OR , pyndmyl, aryl, , , or
(CH~)XOR~
N
~J
wherein x is from 0 to 6, and wherein R1 is independently
selected as defined above. These are also merely representative examples of
the definitions of these substitutents, and are not intended to be exclusive.
Representative compounds in accordance with the present
invention are presented in Table I. This table is not intended to be exclusive
of
the compounds of the present invention, but rather exemplary of the triazine
compounds that are encompassed by this invention.
TABLE I
COMPOUND COMPOUND NAME
NUM)3ER
I NZ-(4-bromo-I-naphthyl) N4-cycloheptyl- -[(I-ethyl-2-
yrrolidinyl)methyl)-1,3,5-triazine-2,4,6-triamine
N'-(4-chloro-I-naphthyl) lV''-cycloheptyl
lN-[(I-ethyl-2-
yrrolidinyl)methyl)-1,3,5-triazine-2,4,6-triamine
3 N -cycloheptyl- -[(I-ethyl-2-pyrrolidinyl)methyl)-
-(3-quinolinyl)-
1,3,5-triazine-2,4,6-triamine
4 NZ-cycloheptyl-N4-[(1-ethyl-2-pyrrolidinyl)methyl]
IV6-(6-quinolinyl)-
1,3,5-triazine-2,4,6-triamine
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COMPOUND COMPOUND NAME
NUMBER
N-cycloheptyl-N4-[(1-ethyl-2-pyrrolidinyl)methyl]-N
-(~ 8-quinolinyl)-
1,3,5-triazine-2,4,6-triatnine
6 N'-cycloheptyl-N4-[(I-ethyl-2-pyrrolidinyl)methyl]-lV-[1-(2-
na hthyl)ethyl]-I,3,5-triazine-2,4,6-triamine
N'-cycloheptyl-N''-(3,4-dichlorophenyl)-N'-[(1-ethyl-2-
pyrrolidinyf)methyl]-1,3,5-triazine-2,4,6-triamine
g N'-cycloheptyl-N~-(3,4-difluorophenyl) N-[(1-ethyl-2-
pyrrolidinyl)methyl]-I ,3,5-triazine-2,4,6-triamine
g N'-cycloheptyl-N''-[(1-ethyl-2-pyrrolidinyl)methyl]-N-[4-
(trifluoromethoxy) heny1 -1,3,5-triazine-2,4,6-triamine
NZ-cycloheptyl-N4-[(I-ethyl-2-pyrrolidinyl)methyl]
IV6-(4-
fluoro henyl)-1,3,5-triazine-2,4,6-triamine
I 1 4-[(4-(cycloheptylamino)-6-{ [( 1-ethyl-2-pyrrolidinyl)methyl]amino}-
1,3,5-triazin-2-yl)-amino]benzonitrile
12 NZ-(4-chlorophenyl)-N4-cycloheptyl-1V6-[(1-ethyl-2-
pyrrolidinyl)methyl]-1,3,5-triazine-2,4,6-triamine
13 NZ-(4-bromophenyl)-N4-cycloheptyl-N6-[(1-ethyl-2-
pyrrolidinyl)methyl]-1,3,5-triazine-2,4,6-triamine
14 Ethyl4-[(4-(cycloheptylamino)-6-{[(I-ethyl-2-
yrrolidinyl)methyl]amino}-1,3,5-triazin-2-yl)-amino]benzoate
N'-(l,l'-biphenyl-4-yl)-N"-cycloheptyl N-[(1-ethyl-2-
yrrolidinyl)methyl]-1,3,5-triazine-2,4,6-triamine
16 N'-cycloheptyllV4-[(I-ethyl-2-pyrrolidinyl)methyl]-1V6-(3-
fluorophenyl)-1,3,5-triazine-2,4,6-triamine
N'-(3-ch l oropheny l)-N4-cyc l oheptyl lVb-[(
1-ethyl-2-
yrrolidinyl)methyl]-1,3,5-triazine-2,4,6-triamine
1g N'-(3-bromophenyl) N'-cycloheptyl-N-[(1-ethyl-2-
yrrolidinyl)methyl]-1,3,5-triazine-2,4,6-triamine
19 Ethyl 3-[(4-(cycloheptylamino)-6- f [(I-ethyl-2-
pyrrol id i ny l)methyl] amino }-1,3,5-triazin-2-yl)-amino]benzoate
N'-cycloheptyl-N4-[(1-ethyl-2-pyrrolidinyl)methyl]
IV6-(2-
fluorophenyl)-1,3,5-triazine-2,4,6-triamine
21 N'-(2-chlorophenyl)-N~-cycloheptyllV-[(1-ethyl-2-
rrolidin I)meth 1]-1,3,5-triazine-2,4,6-triamine
22 NZ-(2-bromophenyl)-N4-cycloheptyl-lVb-[(1-ethyl-2-
pyrrolidinyl)methyl]-1,3,5-triazine-2,4,6-triamine
23 N'-(1,3-benzodioxol-5-yl)-lV''-cycloheptyl
N-[(I-ethyl-2-
yrrolidinyl)meth 1]-1,3,5-triazine-2,4,6-triamine
24 NZ-cycloheptyl IV4-(2,3-dihydro-1,4-benzodioxin-6-yl)
1V6-[(1-ethyl-2-
yrrolidinyl)methyl]-1,3,5-triazine-2,4,6-triamine
NZ-cycloheptyl 1V4-[4-(dimethylamino)phenyl]
1V6-[(1-ethyl-2-
pyrrolidinyl)methyl]-I,3,5-triazine-2,4,6-triamine
26 N'-[3-chloro-4-(diethylamino)phenyl]-1V''-cyclohepty111~-[(1-ethyl-2-
yrrolidinyl)methyl]-1,3,5-triazine-2,4,6-triamine
2~ NZ-cycloheptyl-N4-[(1-ethyl-2-pyrrolidinyl)methyl]-.N6-[4-(4-
mor holinyl) henyl]-1,3,5-triazine-2,4,6-triamine
2g N'-cycloheptyl-N4-[(I-ethyl-2-pyrrolidinyl)methyl]-1V6-[4-(4-methyl-1-
piperazinyl)phenyl]-1,3,5-triazine-2,4,6-triamine
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COMPOUND COMPOUND NAME
NUMBER
N-{4-[(4-(cycloheptylamino)-6-{[(1-ethyl-2-
29 pyrrolidinyl)methyl]amino}-1,3,5-triazin-2-yl)-
amino] henyl acetamide
N-{3-[(4-(cycloheptylamino)-6-{ [( 1-ethyl-2-
30 pyrrolidinyl)methyl]amino}-1,3,5-triazin-2-yl)-
amino] henyl acetamide
31 NZ-cycloheptyl-N4-[(1-ethyl-2-pyrrolidinyl)methyl]
N-(3-
methoxyphenyl)-1,3,5-triazine-2,4,6-triamine
32 NZ-cycloheptyl-IV4-(4-ethoxyphenyl)-IV6-[(1-ethyl-2-
yrrolidinyl)methyl]-I,3,5-triazine-2,4,6-triamine
33 NZ-cYcloheptyl-N4-[(I-ethyl-2-pyrrolidinyl)methyl]
1V-[4-
(methylthio)phenyl]-1,3,5-triazine-2,4,6-triamine
34 N -cycloheptyl-N4-[(1-ethyl-2-pyrrolidinyl)methyl]-1V"-(2-pyridinyl)-
1,3,5-triazine-2,4,6-triamine
35 NZ-cYcloheptyl- -[(1-ethyl-2-pyrrolidinyl)methyl]
IV6-(2-
methylphenyl)-1,3,5-triazine-2,4,6-triamine
36 NZ-cYcloheptyl-N4-[(1-ethyl-2-pyrrolidinyl)methyl]
N-(4-
phenoxyphenyl)-1,3,5-triazine-2,4,6-triamine
3~ NZ-cycloheptyl-N4-[(1-ethyl-2-pyrrolidinyl)methyl]-N-(3-
meth 1 hen 1)-1,3,5-triazine-2,4,6-triamine
3g Na-cycloheptyl lV4-[(I-ethyl-2-pyrrolidinyl)methyl]
N-(4-
methyl henyl)-1,3,5-triazine-2,4,6-triamine
39 2-[(4-(cycloheptylamino)-6-{[(1-ethyl-2-pyrrolidinyl)methyl]amino}-
1,3,5-triazin-2-yl)-amino]-4-methyl-3-thio
henecarboxamide
40 NZ-(4-chlorophenyl)-lV4-cycloheptyllVb-[(1-ethyl-2-
yrrolidinyl)methyl]-NZ-methyl-1,3,5-triazine-2,4,6-triamine
4I 3-[(4-(cycloheptylamino)-6-{ [( 1-ethyl-2-pyrrolidinyl)methyl]amino}-
1,3,5-triazin-2-yl)-(phenyl)amino] ro anenitrile
42 NZ-cYcloheptyl-N4-[(1-ethyl-2-pyrrolidinyl)methyl]
IV-(4-
methoxyphenyl) lV6-methyl-1,3,5-triazine-2,4,6-triamine
43 NZ-cYcloheptyl N'-(2,4-difluorophenyl) N-[(1-ethyl-2-
yrrolidin I)meth 1]-N4-methyl-1,3,5-triazine-2,4,6-triamine
44 [(4-(cYcloheptylamino)-6-{[(I-ethyl-2-pyrrolidinyl)methyl]amino}-
1,3,5-triazin-2-yl)(phenyl)amino]acetonitrile
45 NZ-(3-chlorophenyl)-N"-cycloheptyl-lV-[(1-ethyl-2-
p rrolidinyl)methyl] NZ-methyl-1,3,5-triazine-2,4,6-triamine
46 N'-cycloheptyllV4-[(1-ethyl-2-pyrrolidinyl)methyl]-N-methyllV-[2-
(trifluoromethyl) henyl]-1,3,5-triazine-2,4,6-triamine
47 N'-cycloheptyl-N4-[(1-ethyl-2-pyrrolidinyl)methyl]-N-methyl-N-[4-
(trifl uoromethoxy)phenyl]- I ,3,5-triazine-2,4,6-triamine
4g N'-(3-chloro-4-methoxyphenyl)-N"-cycloheptyllV-[(1-ethyl-2-
rrolidin 1)meth I -1,3,5-triazine-2,4,6-triamine
N-benzoyl-4-[(4-(cycloheptylamino)-6-{[(1-ethyl-2-
49 pyrrolidinyl)methyl]amino}-1,3,5-triazin-2-yl)-
amino benzenesulfonamide
50 NZ-cYcloheptyl IV4-[(1-ethyl-2-pyrrolidinyl)methyl]
N'-(2-naphthyl)-
I ,3,5-triazine-2,4,6-triamine
51 NZ-ethyl-N''-[(I-ethyl-2-pyrrolidinyl)methyl]-1V-(3-fluoro-4-
methoxy hen I)-1,3,5-triazine-2,4,6-triamine
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COMPOUND COMPOUND NAME
NUMBER
52 NZ-(tert-butyl)-N4-[(I-ethyl-2-pyrrolidinyl)methyl]-N
-(3-fluoro-4-
methoxyphenyl)-1,3,5-triazine-2,4,6-triamine
53 NZ-benzyl-N4-[(1-ethyl-2-pyrrolidinyl)methyl]-
-(3-fluoro-4-
methoxy henyl)-1,3,5-triazine-2,4,6-triamine
54 N'-cyclooctyl-N'-[(1-ethyl-2-pyrrolidinyl)methyl]-1V-(3-fluoro-4-
methoxyphenyl)-1,3,5-triazine-2,4,6-triamine
55 NZ-cyclohexyl-N4-[(1-ethyl-2-pyrrolidinyl)methyl]-
-(3-fluoro-4-
methoxyphenyl)-I ,3,5-triazine-2,4,6-triamine
56 N'-cyclopentyl-1V''-[(1-ethyl-2-pyrrolidinyl)methyl]-N''-(3-fluoro-4-
methoxy henyl)-1,3,5-triazine-2,4,6-triamine
N'-[(1-ethyl-2-pyrrolidinyl)methyl] 1V"-(3-fluoro-4-methoxyphenyl)-6-
(1- yrrolidinyl)-1,3,5-triazine-2,4-diamine
Sg Nz-[(I-ethyl-2-pyrrolidinyl)methyl]- -(3-fluoro-4-methoxyphenyl)-
-
hexahydro-1 H-azepin-I-yl-1,3,5-triazine-2,4-diamine
59 N'-[(1-ethyl-2-pyrrolidinyl)methyl]-N"-(3-fluoro-4-methoxyphenyl)-
-
octahydro-I (2H)-quinolinyl-1,3,5-triazine-2,4-diamine
60 NZ-[(1-ethyl-2-pyrrolidinyl)methyl] N'-(3-fluoro-4-methoxyphenyl)-
-
(4-methylcyclohexyl)-1,3,5-triazine-2,4,6-triamine
61 N'-(1-ethyl-pyrrolidin-2-ylmethyl]-N4-(3-fluoro-4-methoxyphenyl)-6-
((S)-2-methoxymethyl-pyrrol id in-1-yl)-1,3,5-triazine-2,4-diamine
62 N'-[(1-ethyl-2-pyrrolidinyl)methyl]-1V"-(3-fluoro-4-methoxyphenyl)-6-
(4-methyl-1-pi erazinyl)-1,3,5-triazine-2,4-diamine
63 6-(4-acetyl-1-piperazinyl) NZ-[(1-ethyl-2-pyrrolidinyl)methyl]-
-(3-
fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4-diamine
64 Ethyl 4-{4-{[(I-ethyl-2-pyrrolidinyl)methyl]amino}
lV''-[(3-fluoro-4-
methoxyphenyl)amino]-1,3,5-triazin-2-yl}-I-
i erazinecarbox late
65 N'-(cyclohexylmethyl) N'-[(I-ethyl-2-pyrrolidinyl)methyl]
IV6-(3-
fluoro-4-methoxy henyl)-1,3,5-triazine-2,4,6-triamine
66 N'-[(1-ethyl-2-pyrrolidinyl)methyl]- -(3-fluoro-4-methoxyphenyl)-
-
(2-furylmethyl)-1,3,5-triazine-2,4,6-triamine
67 N'-[(I-ethyl-2-pyrrolidinyl)methyl]-IV4-(3-fluoro-4-methoxyphenyl)
IV6-
(2,2,2-trifluoroethyl)-1,3,5-triazine-2,4,6-triamine
6g N'-[2-(dimethylamino)ethyl] IV~-[(I-ethyl-2-pyrrolidinyl)methyl]-IV6-
(3-
fluoro-4-methox henyl)-1,3,5-triazine-2,4,6-triamine
N'-[(1-ethyl-2-pyrrolidinyl)methyl] IV4-(3-fluoro-4-methoxyphenyl)-
-
.
69 {4-[2-oxo-2-(1-pyrrolidinyl)ethyl]-I-piperazinyl}-1,3,5-triazine-2,4-
diamine
N',N''-bis[(1-ethyl-2-pyrrotidinyl)methyl]
N5-(3-fluoro-4-
methoxy henyl)-1,3,5-triazine-2,4,6-triamine
~I N'-[(1-ethyl-2-pyrrolidinyl)methyl]-N'-(3-fluoro-4-methoxyphenyl)-Ns
[2-(1- i eridinyl)eth 1 -1,3,5-triazine-2,4,6-triamine
N-[4-(1,3-benzodioxol-5-ylmethyl)-I-piperazinyl]
1Vz-[(I-ethyl-2-
~2 pyrrolidinyl)methyl]-N4-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4-
diamine
~3 N'-[(I-ethyl-2-pyrrolidinyl)methyl] lV4-(3-fluoro-4-methoxyphenyl)-
-
[4-(2- yridinyl)-I-pi erazinyl]-1,3,5-triazine-2,4-diamine
1-[3-( {4-{ [( 1-ethyl-2-pyrrolidinyl)methyl]amino}-6-[(3-fluoro-4-
~4 methoxyphenyl)amino)-1,3,5-triazin-2-yl}amino)propyl]-2-
rrolidinone
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COMPOUND COMPOUND NAME
NUMBER _
NZ-[(1-ethyl-2-pyrrolidinyl)methyl]- -(3-fluoro-4-methoxyphenyl)-
-
[3-(IH-imidazol-1-yl) ro y1 -1,3,5-triazine-2,4,6-triamine
76 Nz-cycloheptyl-N4-ethyl-7V~-(3-fluoro-4-methoxyphenyl)-1,3,5-
triazine-
2,4,6-triamine
N'-(tert-butyl)-lV'"-cycloheptyl-N"-(3-fluoro-4-methoxyphenyl)-1,3,5-
triazine-2,4,6-triamine
~g NZ-benzyl-N4-cycloheptyl-NG-(3-fluoro-4-methoxyphenyl)-1,3,5-
triazine-2,4,6-triamine
1V -cycloheptyl-N'-cyclooctyl N"-(3-fluoro-4-methoxyphenyl)-1,3,5-
triazine-2,4,6-triamine
g0 N'-cycloheptyl-1V''-cyclohexyl-IV-(3-fluoro-4-methoxyphenyl)-1,3,5-
triazine-2,4,6-triamine
g I NZ-cycloheptyl-1V4-cyclopentyl lVb-(3-fluoro-4-methoxyphenyl)-1,3,5-
triazine-2,4,6-triamine
g2 N'-cycloheptyl-N"-(3-fluoro-4-methoxyphenyl)-6-(I-pyrrolidinyl)-
1,3,5-
triazine-2,4-diamine
g3 NZ-cycloheptyl-1V4-(3-fluoro-4-methoxyphenyl)-6-hexahydro-1H-
azepin-1-yl-1,3,5-triazine-2,4-diamine
g4 N'-cycloheptyllV''-(3-fluoro-4-methoxyphenyl)-6-octahydro-I(2H)-
qui nol inyl-1,3,5-triazine-2,4-d f amine
g5 N'-cycloheptyl N'-(3-fluoro-4-methoxyphenyl)
N-(4-
methylc clohexyl)-1,3,5-triazine-2,4,6-triamine
g6 N'-cycloheptyl-N4-(3-fluoro-4-methoxyphenyl)-6-[(2S)-2-
(methoxymethyl)-1-pyrrolidin 1]-1,3,5-triazine-2,4-diamine
g~ N'-cycloheptyllV''-(3-fluoro-4-methoxyphenyl)-6-(4-methyl-1-
piperazinyl)-1,3,5-triazine-2,4-diamine
gg 6-(4-acetyl-1-piperazinyl)-N'-cycloheptyl-IV''-(3-fluoro-4-
methox hen 1)-1,3,5-triazine-2,4-diamine
g9 ethyl-4-{4-(cycloheptylamino)-6-[(3-fluoro-4-methoxyphenyl)amino]-
1,3,5-triazin-2- 1}-1- f erazinecarbox late
90 N'-cycloheptyl IV''-(cyclohexylmethyl) IV6-(3-fluoro-4-
methoxyphenyl)-
1,3,5-triazine-2,4,6-triamine
~I N'-cycloheptyl-N'-(3-fluoro-4-methoxyphenyl)
lV''-(2-furanylmethyl)-
1,3,5-triazine-2,4,6-triamine
92 Nz-cyclohepty11V4-(3-fluoro-4-methoxyphenyl)-
-(2,2,2-
trifluoroethyl)-1,3,5-triazine-2,4,6-triamine
93 N'-cycloheptyl N'-[2-(dimethylamino)ethyl]--N"-(3-fluoro-4-
methoxyphenyl)-1,3,5-triazine-2,4,6-triamine
94 N'-cycloheptyl-N''-(3-fluoro-4-methoxyphenyl)-6-{4-[2-oxo-(I-
yrrolidinyl)ethyl]-1- f erazinyl}-1,3,5-triazine-2,4-diamine
95 N2-cycloheptyl-N -[(1-ethyl-2-pyrrolidinyl)methyl)-
-(3-fluoro-4-
methoxy henyl)-1,3,5-triazine-2,4,6-triamine
96 N'-cycloheptyl-N'-(3-fluoro-4-methoxyphenyl)
N~-[2-(1-
i eridinyl)ethyl]-1,3,5-triazine-2,4,6-triamine
6-[4-(1,3-benzodioxol-5-ylmethyl)1-piperazinyl)
NZ-cycloheptyl N4-(3-
fluoro-4-methoxyhenyl)-I,3,5-triazine-2,4-diamine
9g N'-cycloheptyl-N4-(3-fluoro-4-methoxyphenyl)-6-[4-(2-pyridinyl)-1-
i perazinyl]-1,3,5-tri azine-2,4-triamine
24
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COMPOUND COMPOUND NAME
NUMBER
1-[3-({4-(cycloheptylamino)-6-[(3-fluoro-4-methoxyphenyl)amino]-
1,3,5-triazin-2-yl amino) ro y1]-2- rrolidinone
100 NZ-cycloheptyl-N4-(3-fluoro-4-methoxyphenyl)-
-[3-(IH-imidazol-1-
yl) ro yl]-1,3,5-triazine-2,4,6-triamine
101 (3-Chloro-4-methoxy-phenyl)-(4,6-dichloro-[1,3,5]triazin-2-yl)-amine
102 6-Chforo-N-(3-chioro-4-methoxy-phenyl) N'-cyclohexylmethyl-
[1,3,5]triazine-2,4-diamine
103 N (3-Chloro-4-methoxy-phenyl)-N'-cyclohexyhnethyl
N"-methyl-N"-
(1-methyl-pi eridin-4-yl)-[1,3,5 triazine-2,4,6-triamine
104 6-Chloro N (3-chloro-4-methoxy-phenyl)-N'-(1-propyl-butyl)-
[ 1,3,5]triazine-2,4-diamine
105 N (3-Chloro-4-methoxy-phenyl) N'-methyl IV'-(1-methyl-piperidin-4-
yl)-IV"-(1-prop 1-butyl)-[1,3,5]triazine-2,4,6-triamine
106 N (3-Chloro-4-methoxy-phenyl)-N'-isopropyl
N"-methyl N"-(1-methyl-
piperidin-4-yl)- 1,3,5]triazine-2,4,6-triamine
107 Nz-(3-chloro-4anethoxy-phenyl)- -isopropyl-
-methyl- -piperidin-
4-yl-1,3,5-triazine-2,4,6-triamine
108 5-{4-(3-Chloro-4-methoxy-phenylamino)-6-[methyl-(1-methyl-
i eridin-4-yl)-amino]-[1,3,5 triazin-2-ylamino~-
entan-1-of
109 5-[4-(3-chloro-4-methoxy-phenylamino)-6-(methyl-piperidin-4-yl-
amino)-1,3,5-triazin-2-ylamino - entan-1-of
1 I0 N Butyl-6-chloro N'-(3-chloro-4-methoxy-phenyl)-N-propyl-
[1,3,5]triazine-2,4-diamine
1 I 1 N Butyl-N'-(3-chloro-4-methoxy-phenyl)-N"-methyl-N"-(1-methyl-
piperidin-4-yl) N propyl-[1,3,5]triazine-2,4,6-triamine
I 12 1V -Butyl-N'-(3-chloro-4-methoxy-phenyl)-N-methyl-lV~-piperidin-4-yl-
NZ- ro yl-1,3,5-triazine-2,4,6-triamine
113 2,4-Dichloro-6-cyclohexylmethoxy-[1,3,5]triazine
114 (4-Chloro-6-cyclohexylmethoxy-[1,3,5]triazin-2-yl)-(3-fluoro-4-
methoxy-phenyl)-amine
115 6-Cyclohexylmethoxy-N,N'-bis-(3-fluoro-4-methoxy-phenyl)-1,3,5-
triazine-2,4-diamine
116 6-Cyciohexylmethoxy N (1-ethyl-pyrrolidin-2-ylmethyl)
N'-(3-fluoro-
4-methoxy-phenyl)-[ 1,3,5]triazine-2,4-diamine
117 (4-Chloro-6-cyclohexylmethoxy-[1,3,5]triazin-2-yl)-(3-chloro-4-
methoxy-phenyl)-amine
118 N~'-Bis-(3-chloro-4-methoxy-phenyl)-6-cyclohexylmethoxy-1,3,5-
triazine-2,4-diamine
119 N (3-Chloro-4-methoxy-phenyl)-6-cyclohexylmethoxy
N'-methyl N'-
(1-methyl-piperidin-4-yl)-[1,3,5]triazine-2,4-diamine
120 6-Chloro N,N"-bis-(3-chloro-4-methoxy-phenyl)-[1,3,5]triazine-2,4-
diamine
121 N~'-Bis-(3-chloro-4-methoxy-phenyl) N"-methyl
1V"-(1-methyl-
i eridin-4-yl)-[1,3,5 triazine-2,4,6-triamine
122 N,N'-Bis-(3-chloro-4-methoxy-phenyl)-N"-cycloheptyl-[1,3,5]triazine-
2,4,6-triamine
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COMPOUND COMPOUND NAME
NUMBER
123 N (3-Bromo-4-methoxy-phenyl)-N'-cycloheptyl-N"-methyl-N"-(I-
methyl-pi eridin-4-yl)- 1,3,5]triazine-2,4,6-triamine
124 (4,6-Dichloro-[1,3,5]triazin-2-yl)-(3-fluoro-4-methoxy-phenyl)-amine
125 6-Chloro N cyclohexylmethyl N'-(3-fluoro-4-methoxy-phenyl)-
[1,3,5]triazine-2,4-diamine
126 N Cyclohexylmethyl-N'-(1-ethyl-pyrrolidin-2-ylmethyl)-.N"-(3-fluoro-
4-methoxy-phenyl)-[1,3,5]triazine-2,4,6-triamine
127 6-Chloro-N-cycloheheptyl-N'-(3-fluoro-4-methoxy-phenyl)-
[ 1,3,5]triazine-2,4-diamine
128 N Cycloheptyl-N'-(3-fluoro-4-methoxy-phenyl)-6-pyrrolidin-1-yl-
[ 1,3,5]triazine-2,4-diamine
129 N-Cycloheptyl-N'-ethyl-N"-(3-fluoro-4-methoxy-phenyl)-
[ 1,3,5]triazine-2,4-diamine
130 N-Cycloheptyl-N'-(1-ethyl-pyrrolidin-2-ylmethyl)
N"-(3-fluoro-4-
methoxy- henyl)-[1,3,5]triazine-2,4,6-triamine
131 2-[4-chloro-6-(3-chloro-4-methoxy-phenylamino)-[1,3,5]triazin-2-
ylamino]-propane-1,3-diol
132 2-{4-(3-chloro-4-methoxy-phenylamino)-6-[methyl-(1-methyl-
pi eridin-4-yl)-amino]-[1,3,5]triazin-2-
lamino - ro ane-1,3-diol
133 6-Chloro-N (3-chloro-4-methoxy-phenyl) N-cycloheptyl-
[1,3,5]triazine-2,4-diamine
134 N (1-benzyl-piperidin-4-yl) N'-(3-chloro-4-methoxy-phenyl)
1V"-
cyclohe tyl-[1,3,5]-2,4,6-triamine
135 NZ-(3-chloro-4-methoxy-phenyl)-N4-cycloheptyl-1V4-piperidin-4-yl-
1,3,5-triazine-2,4,6-triamine
136 Nz-(3-chloro-4-methoxy-phenyl)-N"-cycloheptyl-N'-(1-ethyl-pyrrolidin-
2-ylmethyl)-1,3,5-triazine-2,4,6-triamine
137 N (3-Chioro-4-methoxy-phenyl) N'-cycloheptyl-N'-methyl
~V"-(1-
methyl-piperidin-4-yl)-[ 1,3,5]triazine-2,4,6-triamine
138 2-chloro-4- f 4-cycloheptylamino-6-[methyl-(I-methyl-piperidin-4-yl-
asnino]-1,3,5-triazin-2-ylamino}-phenol
139 Nz-cycloheptyl-N-((S)-I-ethyl-pyrrolidin-2-ylmethyl)-1V-(3-fluoro-4-
methoxy hen I)-1,3,5-triazine-2,4,6-triamine
l40 N'-cycloheptyl-N4-((R)-1-ethyl-pyrrolidin-2-ylmethyl)-.IV-(3-fluoro-4-
methoxyphenyl)-1,3,5-triazine-2,4,6-triamine
141 IVz-cyclohexylmethyl-N"-((S)-I-ethyl-pyrrolidin-2-ylmethyl)
N-(3-
fluoro-4-methox henyl)-1,3,5-triazine-2,4,6-triamine
142 N'-cyclohexylmethyl-N4-((R)-1-ethyl-pyrrolidin-2-ylmethyl)
11~"-(3-
fluoro-4-snethox henyl)-I,3,5-triazine-2,4,6-triamine
42, Scheme 23)
143 (f 4-cycloheptylamino-6-[((S~-I-ethyl-pyrrolidin-2-ylmethyl)-amino]-
1,3,5-triazin-2-yl}-phenyl-amino)-acetonitrile
(43, Scheme 24)
144 ({4-cycioheptylamino-6-[((R)-I-ethyl-pyrrolidin-2-ylmethyl)-amino]-
1,3,5-triazin-2- 1}- hen 1-amino)-acetonitrile
145 Nz-[(1-ethyl-2-pyrrolidinyl]-N"-(3-fluoro-4-methoxyphenyl)-6-[(5~-2-
(methoxymethyi)-I- yrrofidin 1 -1,3,5-triazine-2,4-diamine
146 Nz-(3-chloro-4-methoxy-phenyl) 7V4-cycloheptyl
IV"-methyl N-
piperidin-4-yl-1,3,5-triazine-2,4,6-triamine
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COMPOUND COMPOUND NAME
NUMBER
147 4-(3-Chloro-4-methoxy-phenylamino)-6-cycloheptylamino-1,3,5-
triazin-2-of
148 N (I-Aza-bicyclo[2.2.2]oct-3-yl)-N'-(3-chloro-4-methoxy-phenyl)
N"-
)1-ethyl- yrrolidin-2-yhnethyl)-[1,3,5]triazine-2,4,6-triamine
149 N'-(3-chloro-4-diethylamino-phenyl)-lV''-cycloheptyl-N'-(1-ethyl-
yrro I id i n-2-y I methyl)-1,3,5-triazine-2,4,6-triamine
150 NZ-cycloheptyl-N4-(2-dimethylamino-ethyl)
IV6-(3-fluoro-4-methoxy-
phenyl)-1,3,5-triazine-2,4,6-triamine
151 ({4-cycloheptylamino-6-[I-ethyl-pyrrolidin-2-ylmethyl)-amino]-1,3,5-
triazin-2-yl}- henyl-amino)-acetonitrile
152 N Azepan-1-yl-6-chloro-N'-(3-chloro-4-methoxy-phenyl)-
[1,3,5]triazine-2,4-diamine
153 N"-(3-chloro-4-methoxy-phenyl)-N,N'-bis-perhydro-azepin-1-yl-1,3,5-
triazine-2,4,6-triamine
154 N Azepan-I-yl-N'-(3-chloro-4-methoxy-phenyl)
N"-(1-methyl-
pi eridin-4-yl)-[1,3,5]triazine-2,4,6-triamine
155 N~-(3-chloro-4-methoxy-phenyl)-1V6-methyl-NZ-perhydro-azepin-1-yl-
N6-piperidin-4-yl-1,3,5-triazine-2,4,6-triamine
156 N~'-di-n-propyl N"-(3-fluoro-4-methoxy-phenyl)-1,3,5-triazine-2,4,6-
triamine
157 N~'-dicyclopropyl-N"-(3-fluoro-4-methoxy-phenyl)-1,3,5-triazine-
2,4,6-triamine
158 NZ-Cycloheptyl-N''-(3-fluoro-4-methoxy-phenyl)
N6-methyl IV6-(1-
methyl-piperidin-4-yl)-1,3,5-triazine-2,4,6-triamine
159 N'-Cycloheptyl-N4-(3-fluoro-4-methoxy-phenyl)-1V6-methyllVb-
iperi d i n-4-yl- I ,3,5-triazine-2,4,6-triamine
N'-cycloheptyl-N'-(3-fluoro-4-methoxyphenyl)
N-methyl-N-(1-
160 methyl-piperidin-4-yl)-1,3,5-triazine-2,4,6-triamine,
hydrogen chloride
salt
[N (3-Chloro-4-methoxy-phenyl)-N'-cycloheptyl
IV"-methyl N"-(1-
161 methyl-piperidin-4-yl)-[1,3,5]trizaine-2,4,6-triamine,
hydrogen chloride
salt
162 N'-(3-chloro-4-diethylamino-phenyl)-N"-cycloheptyl-N-(1-ethyl-
yrrolidin-2-ylmethyl)-1,3,5-triazine-2,4,6-triamine
N'-(3-chloro-4-diethylamino-phenyl)-N''-cycloheptyl
N-(1-ethyl-
163 pyrrolidin-2-ylmethyl)-1,3,5-triazine-2,4,6-triamine
hydrogen chloride
salt
164 N'-cycloheptyl 1V4-(I-ethyl-pyrrolidin-2-ylmethyl)
IV6-(3-fluoro-4-
methoxy henyl)-1,3,5-triazine-2,4,6-triamine
hydro en chloride salt
N'-(cyclohexyhnethyl) N'-[(I-ethyl-2-pyrrolidinyl)methyl]-N-(4-
165 fluoro-3-methoxyphenyl)-1,3,5-triazine-2,4,6-triamine
hydrogen
chloride salt
166 ({4-cycloheptylamino-6-[(1-ethyl-pyrrolidin-2-ylmethyl)-amino]-1,3,5-
triazin-2-yl}-phenyl-amino)-acetonitrile
h dro en chloride salt
167 N~-cycloheptyl-N4-(3-fluoro-4-methoxy-phenyl)
N~-methyl-1V6-(1-
methyl-piperidin-4-yl)-1,3,5-triazine-2,4,6-triamine
maleate salt
168 NZ-cycloheptyl-N4-(3-fluoro-4-methoxy-phenyl)-N6-methyllVb-(1-
methyl- iperidin-4-yl)-1,3,5-triazine-2,4,6-triamine
citrate salt
27
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COMPOUND COMPOUND NAME
NUMBER
169 NZ-cycloheptyl-N4-(3-fluoro-4-methoxy-phenyl)-IVY-methyl-
-(1-
methyl- i eridin-4-yl)-1,3,5-triazine-2,4,6-triamine
succinate salt
N (3-Bromo-4-methoxy-phenyl)-N'-cycloheptyl-N"-methyl-N"-(1-
170 methyl-piperidin-4-yl)-[1,3,5]triazine-2,4,6-triamine
hydrogen chloride
salt
1n general terms, the compositions in accordance with the present
invention also comprise tris(amino) triazine compounds of the following
structure:
R~-N
-N Rs
N ~~-N-R~.
>=N
R6-N
Rs
wherein R~ to R6 represent H, alkyl, aryl, alkenyl, alkynyl, aralkyl,
aralkenyl,
aralkynyl, cycloalkyl, cycloalkenyl, heteroalkyl, heteroaryl, halide, alkoxy,
aryloxy, alkylthio, arylthio, silyl, siloxy, amino, alkylamino, dialkylamino
and
the like, including straight or branched chain derivatives thereof, cyclic
derivatives thereof, substituted derivatives thereof, heteroatom derivatives
thereof, heterocyclic derivatives thereof, functionalized derivatives thereof,
salts
thereof, isomers thereof, or combinations thereof.
For example, a typical substituent R~ to R6 is a substituted alkyl, in
which the substituent is a heterocyclic derivative. Examples of nitrogen
containing heterocyclic moieties include, but are. not limited to groups such
as
pyridinyl (derived from pyridine, bonded through a ring carbon), piperidinyl
(derived from piperidine and bonded through the ring nitrogen atom or a ring
carbon), and pyrrolidinyl (derived from pyrrolidine and bonded through the
ring
nitrogen atom or a ring carbon).
Examples of substituted or functionalized derivatives of RI to R6
include, but are not limited to, moieties containing substituents such as
acyl,
formyl, hydroxy, acyl halide, amide, amino, azido, acid, alkoxy, aryloxy,
halide,
carbonyl, ether, ester, thioether, thioester, nitrile, alkylthio, arylthio,
sulfonic acid
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and salts thereof, thiol, alkenyl, alkynyl, nitro, imine, imide, alkyl, aryl,
combinations thereof, and the like. Moreover, in the case of alkylated
derivatives
of the recited moieties, the alkyl substitutent may be pendant to the recited
chemical moiety, or used for bonding to the amine nitrogen through the alkyl
substituent.
Examples of chemical moieties R~ to Rb of the present invention
further include, but are not limited to: H; methyl; ethyl; propyl; butyl;
pentyl;
hexyl; heptyl; octyl; ethenyl; propenyl; butenyl; ethynyl; propynyl; butynyl;
cyclobutyl; cyclopentyl; cyclohexyl; cyclobutenyl; cyclopentenyl;
cyclohexenyl;
phenyl; tolyl; xylyl; benzyl; naphthyl; pyridinyl; furanyl; tetrahydro-1-
napthyl;
piperidinyl; indolyl; indolinyl; pyrrolidinyl; 2-(methoxymethyl) pyrrolidinyl;
piperazinyl; quinolinyl; quinolyl; alkylated-1,3-dioxolane; triazinyl;
morpholinyl;
phenyl pyrazolyl; indanyl; indonyl pyrazolyl; thiadiazolyl; rhodaninyl;
thiolactonyl; dibenzofuranyl; benzothiazolyl; homopiperidinyl; thiazolyl;
quinonuclidinyl; isoxazolidinonyl; any isomers, derivatives, or substituted
analogs thereof; or any substituted or unsubstituted chemical groups such as
alcohol, ether, thiol, thioether, tertiary amine, secondary amine, primary
amine,
ester, thioester, carboxylic acid, diol, diester, acrylic acid, acrylic ester,
methionine ethyl ester, benzyl-I-cysteine ethyl ester, imine, aldehyde,
ketone,
amide, or dime.
Further examples of chemical moieties R~ to R6 of this invention
include, but are not limited to, the following species or substituted or
alkylated
derivatives of the following species, covalently bonded to the amine nitrogen:
furan; tetrahydrofuran; indole; piperazine; pyrrolidine; pyrrolidinone;
pyridine;
quinoline; anthracene; tetrahydroquinoline; naphthalene; pyrazole; imidazole;
thiophene; pyrrolidine; morpholine; and the like. One feature of the recited
species or substituted or alkylated derivatives of these species, is that they
may be
covalently bonded to the amine nitrogen in any fashion, including through the
pendant substituent or alkyl group, through the heteroatom as appropriate, or
through a ring atom as appropriate, as understood by one of ordinary skill in
the
art.
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The chemical moieties R~ to R6 of the present invention also
include, but are not limited to, cyclic alkanes and alkenes and include
bridged and
non-bridged rings. Examples of bridged rings include, but are not limited to,
groups such as norbornyl; norbonadienyl, adamantyl; 6-
azabicyclo[3.2.1]octanyl;
3-azabicyclo(2.2.2]octanyl, and the like.
In one embodiment of the present invention, NR~Rz, NR3R4, or
NRSR6 are derived from a cyclic secondary amine. Examples of a cyclic amino
chemical moiety of the present invention include, but are not limited to
piperidine; 4-benzyl-piperidine; 3-piperidinemethanol; moropholine;
4-piperidinopiperidine; I-(2-amino-methyl)-piperazine; decahydroquinoline;
1,2,3,4-tetrahydro-pyridoindole (either amine moiety); 3-amino-5-phenyl
pyrazole; 3-aminopyrazole; histidinol; hexamethyleimine; 4-hydroxypiperidine;
2-piperidinemethanol; I, 3, 3-trimethyl-6-azabicyclo[3.2.1] octane;
3-pyrrolidinol; 1-methylpiperazine; 2-ethyl-piperidine; I, 2, 3,
4-tetrahydroisoquinoline; 3-aminopyrrolidine; 2, 6-dimethylmorpholine; 2, 3,
4-tetrahydroisoquinoline; 1, 2, 3, 4-tetrahydroquinoline; I-(2-methoxyphenyl)
piperazine; 2, 6-dimethylpiperazine (either amine moiety); iminodibenzyl;
5-methoxytryptamine; 4, 4'-bipiperidine; 1-(2-hydroxyethyl) piperazine;
4-methylpiperidine; and the like.
Importantly, the general structure of the present invention
encompasses all states of saturation of the substitutents shown, such as all
ene,
diene, triene, and yne derivatives of any substitutent. The general structure
also
encompasses all conformational isomers, regioisomers, and stereoisomers that
may arise from a particular set of substitutents. The general structure also
encompasses all enantiomers, diastereomers, and other optical isomers whether
in
enantiomeric or racemic forms, or mixtures of stereoisomers.
Preparation of the Focused Library of Compounds
Many of the compounds of this invention were prepared in a
parallel synthetic procedure according to the methods described below.
Examples of compounds prepared by the parallel synthesis techniques are
provided in Table 2. These preparations involve reacting the individual amine
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compounds (monomers) with cyanuric chloride, which are also presented in
Table 2, along with the chemical structures of compounds prepared by the
parallel synthesis methods.
A library of compounds was synthesized according to the present
invention to afford substituted N2, N~, 1V6-tris(amino)-1,3,5-triazines, as
follows.
The design of the compound library was based primarily on structure 95 shown
below. That is, the design of the Nd, Nø, IV6-tris(amino)triazines was focused
so
that only one of the pendant amino groups (NA, NB, or N~ in the structure
above)
was changed during each synthesis, while the other two groups were held
constant. The combination of the specific amines employed produced a library
of
compounds of novel composition. Initially, the library was developed using
methyl-(1-methyl-piperidin-4-yl)-amine, holding the cycloheptyl and m-
fluoroanisidyl groups constant (in structure 95 below). The synthesis of the
triazines around methyl-(I-methyl-piperidin-4-yl)-amine was not optimized, and
I S the amine was subsequently replaced with (I-ethyl-pyrrolidin-2-yl)-
methylamine
which provided a more tractable synthesis.
OCH3 OCH3
HN ~ ~ F HN\~~- ~ ~ ~ HN ~ ~ F
N~N~--N H N~ ~~--N H N~ ~>---N H
H3C~N~N H3C~N~N HN~N
N~ N
CH3 CH3
171 172 95
The library of N~,1V~, 1V6-tris(amino)-1,3,5-triazines was prepared
based on the strategy of changing only one pendant amino group per synthesis,
and based on the parent structure 95 shown above. The library was divided into
three subgroups: Libraries I, 1I, and III (shown in Table 2). Library I
includes
compounds having unchanged NB and N~ groups but different NA groups (6).
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The pendant amino group NA was changed according to the specific examples
listed below. Library II includes compounds having unchanged NA and N~
groups and different NB groups (7). Pendant amino group NB was changed
according to the specific examples listed below. Library III includes
compounds
having unchanged NA and NB groups and different Nc groups (8). The pendant
amino group Nc was changed according to the specific examples listed below.
The NZ, N~', lV~-tris(amino)-1,3,5-triazine compound structures that
are presented in Tables 2 and following were generated using ISIS-DrawTM
version 2.4Ø20, and were generated with the option to display unspecified
hydrogen atoms if not shown, however, not all hydrogen atoms were displayed in
the structures shown. In all structures presented in any text, table, scheme
or
figure herein, any hydrogen atoms that are required for any atom to attain its
usual valence, whether a carbon atom or a heteroatom, should be inferred if it
is
not specifically indicated in a structure.
One method of preparation of the compounds is shown in the
scheme below. The compounds were prepared by reacting cyanuric chloride
sequentially with monomers of primary or secondary amines to afford the
desired
1,3,5-triazine derivatives [1,2,3,4]. Thus, the amine starting compounds that
are
used to react with cyanuric chloride are termed "monomers." The N2, N°,
1V6-
tris(amino-substituted)-1,3,5-triazines were prepared without the need for
purification between each step of the synthesis, and the final product was
isolated
by standard procedures. Purification was accomplished using flash column
chromatography as needed. It is within the skill of the art of organic
synthesis to
prepare, isolate and purify these organic compounds described herein, and to
modify the syntheses shown. For example, it is possible to synthesize the
compounds of the present invention by using an excess of any monomers of
primary or secondary amines in any of the three steps shown in Scheme 1, such
that the excess monomer, serves as both substituent for the triazine core, as
well
as a base, in which case i-PraNEt base can be excluded.
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R1
CI
N R~RZNH, i-Pr2NEt R~- ~N R3RqNH, i-Pr2NEt
/N N CI N~ ~>-CI
N
CI CH3CN/1,4-dioxane CI~ CH3CN/1,4-dioxane
R~ R~
RZ N~N R3 RSR6NH, i-PrZNEt R2 N~N R
~N N_Ra //~N N_Ra
CI CH3CN/1,4-dioxane Rs-N
Rs
The pendant amino groups can be substituted by functional groups
depicted as R~ to R6 groups in Scheme 1. The degree of functionality of a
pendant amino group is determined by the structure and complexity of the amine
monomer, and will affect the overall molecular diversity of the N~,1V~,N6-
tris(amino-substituted)-1,3,5-triazines. A wide range of amine monomers may be
used in this invention. Once bonded to the triazine core, the pendant amino
groups can be described as secondary or tertiary substituted, depending on the
degree of substitution at the nitrogen atom.
Table 2 presents charts of N2,Nø~,N6-tris(amino)-1,3,5-triazine
compounds of Libraries I-III of this invention, respectively, along with the
amine
precursor monomers used in the preparation of the compounds. General
procedures and synthetic procedures are detailed in Example 1-5. The sequence
in which each monomer is added in Scheme 1 is also presented in Table 2, where
Monomer 1 is added first, Monomer 2 added second, and Monomer 3 is added
third. While not intending to be bound by the following statement, it is
believed
that this order of addition is significant, because each synthetic stop
necessarily
involves the reaction of a monomer with a different triazine precursor. That
is,
monomer 1 reacts with cyanuric chloride, monomer 2 reacts with an amino
dichloro(triazine), and monomer 3 reacts with a diamino chloro(triazine), as
shown in Scheme 1. Thus, the order in which the monomers are employed is
based on the general synthetic principle that the relative nucleophilicity
and/or
basicity of monomers 1-3 used in the synthetic scheme should generally
increase
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from monomer 1 to monomer 3. This strategy permits the most nucleophilic
and/or basic amine monomer to be reacted with the more sterically congested
and
presumably less-reactive diamino chloro(triazine), where its greater
reactivity
may assist the reaction proceeding to completion. In some cases, more than one
order of monomer addition will provide the desired product, but the reaction
sequences provided in Table 2 represents the optimum synthetic methods
presently known.
Note that only in a general sense do the substituents indicated as
NA, NB, and No in the general structures above correspond with the actual
N~,1V~,
Ns-nomenclature of the NZ, Nø, 1V6-tris(amino)triazines. Because the order in
which Nz, N~, and 1V6 substituents are assigned a 2-, 4-, or 6-position on the
triazine core is dependent on the name of each amino group in the molecule, it
is
not always true that one particular amino group always appears as an N~, lV~,
or
1V6 substituent, even when only a single substituent is being permuted at one
position. For example, many of the compounds of Table 2 contain both
cycloheptyl amino and 3-fluoro-4-methoxyphenyl amino groups, yet these groups
take on different 2-, 4-, or 6-positions as a function of the name of the
third
substituent on the triazine core. As a result, the syntheses are discussed in
terms
of permuting amino groups at one pendant NA, NB, or N~ position (rather than
NZ,
N~, or 1V6 position) in the structure above, while maintaining the other amino
groups constant. Further, note that the compound names used in the Tables,
Claims and specification were typically generated using Beilstein's Autonomy
4.01.188, as well as the earlier CD "stand-alone" version of Beilstein's
AutonomTM, Autonom 2000. Typically, the compound names generated in this
fashion were used, regardless of whether the compound name is an IUPAC, CAS,
Beilstein, or other nomenclature. In each case however, the names
unambiguously identify the compound specified.
A. Amino Groups Derived from Monomer 1
The sequence of monomer reaction with the triazine core, shown
in Scheme 1, is Monomer l, Monomer 2, and Monomer 3, added in that order.
Thus, an amino dichloro(triazine) is formed from Monomer 1 and cyanuric
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chloride. For the first pendant amino group derived from Monomer 1 and
cyanuric chloride, the Monomer 1 amine used and proposed included primarily,
but not always, aryl amines, specifically phenyl, naphthyl, naphthylallcyl,
quinolinyl, heteroaryl derivatives, and the like.
Specific examples of Monomer 1 used to produce the first pendant
amino group in N', 1V~, 1V~-tris(amino-substituted)-1,3,5-triazines, and their
[Chemical Abstract Registry numbers] include, but are not limited to, 4-
chloroaniline [106-47-9], 3,4-ethylenedioxaniline [22013-33-8], 4-bromoanline
[106-40-1], ethyl 4-aminobenzoate [94-09-7], 4-fluoro-aniline [371-40-4], 4-
aminobiphenyl [92-67-I], 3-fluoroaniline [372-19-0], 2-aminonaphthalene [91-
59-8], 3-chloroaniline [108-42-9], 4-morpholinoaniline [2524-67-6], 3-
bromoaniline [591-19-5], 4'-aminoacetanilide [122-80-5], ethyl 3-aminobenzoate
[582-33-2] nz-aminoacetanilide [102-28-3], 2-fluoroaniline [348-54-9], m-
anisidine [536-90-3], 2-chloroaniline [[95-51-2], p-phenetidine [156-43-4], 2-
bromoaniline [615-36-1], 4-(methylthio)aniline [104-96-I], 3,4-(methylendioxy)
aniline [14268-66-7], 2-aminopyridine [504-29-0], o-toluidine [95-53-4], 2,4-
difluoro-N methylaniline [138564-16-6], 4-phenoxyaniline [139-59-3], N
phenylglycinonitrile [3009-97-0], m-toluidine [108-44-1], 3-chloro N
methylaniline [7006-52-2], p-toluidine 106-49-O], 2-
(methylamino)benzotrifluoride, 4-chloro-N methylaniline [932-96-7], 4-
aminobenzonitrile [873-74-5], 3-anilinopropionitrile, [1075-76-9], tetracaine
[94-
24-6], N methyl p-anisidine [5961-59-1], 3-chloro p-anisidine [5345-54-O],
sulfabenzamide [127-71-9], 3-aminoquinoline [580-17-6], 1-amino-4-
bromonaphthalene [2298-07-9], . 6-aminoquinoline [580-15-4] 1-amino-4-
chloronaphthalene, [4684-12-2] 8-aminoquinoline [578-66-5], S-(-)-1-(2-
naphthyl)-ethylamine [3082-62-0], 3,4-dichloroaniline [95-76-1], 3,4-
difluoroaniline [3863-I 1-4], N methyl-4-(trifluoromethoxy)aniline [41419-59-
4],
4-(trifluoromethoxy)aniline [461-82-5], 2-amino-4-methylthiophene-3-
carboxamide [4651-97-2], N,N diethyl N'-phenethylenediamine [1665-59-4],1-
(4-amino-phenyl)-4-methylpiperazine hydrochloride [94520-33-9], 2-chloro-
N',N'-diethyl-1,4-phenylenediamine monohydrochloride [196938-07-5] 2-
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(dimethylamino)ethyl 4-aminobenzoate [11012-47-2], N,N dimethyl-1,4-
phenylenediamine [1665-95-4].
B. Amino Groups Derived from Monomer 2
The reaction of Monomer 2 with a preformed amino
dichloro(triazine) provides an intermediate diamino chloro(triazine) in the
synthesis of N~, lV~, IVG-tris(amino-substituted)-1,3,5-triazines. Thus, for
bonding
the second pendant amino group to the triazine core, the Monomer 2 amine used
and proposed included amines, specifically alkyl (C~-C~2, straight chain or
branched), cycloalkyl (C3-C1o ring size), azacyclo (CZ-Coo), and benzyl amine
derivatives. The ring of the cycloalkyl and azacycloamine, and phenyl ring of
the benzyl derivatives can be optionally substituted with one or more
moieties, or
a combination of moieties, such as, alkyl, alkenyl, alkynyl, phenyl, benzyl,
halo,
cyano, nitro, hydroxy, thioxy, alkoxy, aryloxy, haloalkyloxy, alkylthio,
arylthio,
I S amino, alkyl amino, aryl amino, acyl, carboxyl, amido, sulfonamido,
sulfonyl,
sulfate, sulfonic acid, morpholino, thiomorpholino, piperazinyl, pyridyl,
thienyl,
furanyl, pyrroyl, pyrazoyl, phosphate, phosphonic acid, phosphonate and the
like.
These groups can be represented in protected or unprotected forms used in
standard organic synthesis.
2p In addition, any monomer described that has a stereocenter
includes its enantiomers, diastereomers, and optical isomers whether in
enantiomeric or racemic forms, or mixtures of stereoisomers. This is to
include
all of the 1,3,5-triazine derivatives and their stereoisomers presented herein
that
are formed as a result.of using optically active, scalemic or racemic
monomers.
25 Specific examples of Monomer 2 used to attach the second
pendant amino group in the synthesis of N~,1V~, IV6-tris(amino-substituted)-
1,3,5-
triazines, and their corresponding [Chemical Abstract Registry numbers]
include,
but are not limited to, ethylamine [75-04-07], cyclohexanemethylamine [3128-
02-8] tart-butylamine [75-64-9], furfurylamine [617-89-0], benzylamine [100-46-
30 9], 2,2,2-trifluroethylamine [753-90-2], cyclooctylamine [5452-37-9N,lV
dimethylethylenediamine cyclohexylamine [108-91-8], cyclopentylamine [1003-
03-8), I-(2-aminoethyl)-piperidine [26116-12-1], 1-acetylpiperazine [13096-96-
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3], pyrrolidine (123-75-I], 1-piperonylpiperazine [32231-06-4],
hexamethyleneimine [111-49-9], 1-(2-pyridyl)piperazine [34803-66-2],
decahydroquinoline (cis/trans) [2051-28-7], 1-methylpiperazine [109-Ol-3], 1-
(3-
aminopropyl)-imidazole [5036-48-6], ethyl I-piperazine carboxylate [120-43-4],
4-methylcyclohexylamine (cis/trans) [6321-23-9], 1-(3-aminopropyl)-2-
pyrrolidine [7663-77-6], 2-(aminomethyl)-ethy-lpyrrolidine [26116-12-1], (+)-S-
2-(methoxymethyl) pyrrolidine [63126-47-6], 1-(pyrrolidineo carbonylmethyl)
piperazine [339890-45-4].
C. Amino Groups Derived from Monomer 3
The reaction of Monomer 3 with a preformed diamino
chloro(triazine) provides the final step in the synthesis of Na, Nø,1V6-
tris(amino-
substituted)-1,3,5-triazines. Thus, for bonding the third pendant amino group
to
the triazine core, the Monomer 3 used and proposed consisted of amines,
I S specifically alkyl (Ci-C,Z, straight chain or branched), cycloalkyl (C3-
Coo ring
size), azacyclo (CZ-Cio), and benzyl amine derivatives. The ring of these
cycloalkyl-, azacycloamine, and phenyl ring of the benzyl derivatives can be
i optionally substituted with one or more moieties, or a combination of
moieties
such as groups as alleyl, alkenyl, alkynyl, phenyl, benzyl, halo, cyano,
nitro,
hydroxy, thioxy, alkoxy, aryloxy, haloalkyloxy, alkylthio, arylthio, amino,
alkyl
amino, aryl amino, acyl, carboxyl, amido, sulfonamido, sulfonyl, sulfate,
sulfonic
acid, morpholino, thiomorpholino, piperazinyl, pyridyl, thienyl, furanyl,
pyrroyl,
pyrazoyl, phosphate, phosphonic acid, phosphonate and the like.
In addition, any monomer described that has a stereocenter
includes its enantiomers, diastereomers, and optical isomers whether in
enantiomeric or racemic forms, or mixtures of stereoisomers. This is to
include
all of the 1,3,5-triazine derivatives and their stereoisomers presented herein
that
are formed as a result of using optically active, scalemic or racemic
monomers.
Specific examples of Monomer 3 used to attach the third pendant
amino group in the synthesis ofNa, N~,1V6-tris(amino-substituted)-1,3,5-
triazines,
and their corresponding [Chemical Abstract Registry numbers] used in the
synthesis of the N', N~, N~-tris(amino-substituted)-1,3,5-triazine derivatives
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include, but are not limited to, ethylamine [75-04-07], cyclohexanemethylamine
[3128-02-08], tent-butylamine [75-64-9], furfurylamine [617-89-0], benzylamine
[100-46-9], 2,2,2-trifluroethylamine [753-90-2], cyclooctylamine [5452-37-9],
N,N dimethylethylenediamine, cyclohexylamine [108-91-8], cyclopentylamine
[1003-03-8], 1-(2-aminoethyl)-piperidine, [26116-12-1], 1-acetylpiperazine
[13096-96-3], pyrrolidine [123-75-1], 1-piperonylpiperazine [32231-06-4],
hexamethyleneimine [III-49-9], 1-(2-pyridyl)piperazine [34803-66-2],
decahydroquinoline (cis/trans) [2051-28-7], 1-methylpiperazine [109-O1-3], 1-
(3-
aminopropyl)-imidazole [5036-48-6], ethyl 1-piperazine carboxylate [120-43-4],
4-methylcyclohexylamine (cis/trans) [6321-23-9], 1-(3-aminopropyl)-2-
pyrrolidine [7663-77-6], 2-(aminomethyl)-ethyl-pyrrolidine [26116-12-1], (+)-S
2-(methoxymethyl) pyrrolidine [63126-47-6], 1-(pyrrolidineoearbonylmethyl)
piperazine [339890-45-4].
In addition to the parallel synthetic procedures used to prepare the
compounds of Table 2, Table 1 also provides other exemplary triazine
compounds of the present invention, which were synthesized individually rather
than using parallel syntheses. The complete preparation and properties of
these
compounds are presented in the Examples, where details of the synthetic
procedures used are provided. The synthetic procedures for these compounds
involve both the substitution of chloride groups on cyanuric chloride, as well
as
various chemical modifications of these groups once bonded to the trizine
core.
In particular, this invention also encompasses salts of the neutral triazine
compounds, as provided in the Examples and the Tables.
In another aspect of this invention, compounds of the present
invention include, but are not limited to, those having the following formula:
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RsN
Y
or an ene, a dime, a triene, or an yne derivative thereof; a saturated
derivative
thereof; a stereoisomer thereof; or a salt thereof;
wherein:
R' is in each occurrence independently selected from H; linear or
branched alkyl with up to 10 carbon atoms; or cycloalkyl with up to 10 carbon
atoms;
X' is selected from m-F, rra-Cl, sn-Br, m-I, m-CN, m-N02, m-
SOZR~, or m-S020R~, or X~ and X2 together is a fused benzene, pyridine, or
dioxane ring;
X~ is selected from p-OR', p-SR', p-NRI2, p-OM, or p-SM,
wherein M is selected from Li, Na, K, Mg, or Ca;
Y' is selected from cycloalkyl with up to 10 carbon atoms; linear
or branched alkyl with up to 10 carbon atoms; CH2Ra, wherein Ra is a
cycloalkyl
~CH2)n
N
I 5 with up to 10 carbon atoms; or ~ , wherein n is 1 or 2;
AYE is selected from a halogen or OR', or
~~CHR~
NJ NR~
A is NR~ and Y2 is selected from R', R~ , or
Compositions comprising compounds of this formula are also
encompassed by the present invention, as well as mixtures or combinations of
compounds of this formula.
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In a further aspect of this invention, compounds of the present
invention include, but are not limited to, those having the following formula:
X1
S
or an ene, a dime, a triene, or an yne derivative thereof; a saturated
derivative
thereof; a stereoisomer thereof; or a salt thereof;
wherein:
R' is in each occurrence independently selected from -H; linear or
branched alkyl with up to 10 carbon atoms; cycloalkyl with up to 10 carbon
atoms; or aryl;
E is CH or N;
n is an integer from 0 to 3;
X' is selected from -H, m-F, m-Cl, m-Br, m-I, m-CN, m-NOa, m-
SOZR', or m-SOZORI, or X' and X~ together is a fused benzene or pyridine ring;
XZ is selected from -H, o-Cl, o-Br, p-OR', p-SRI, p-NRi~, p-F, p-
-C O OR', p-OM, or p-SM, wherein M is selected from Li, Na,
C l, p-B r, p-CF3, p ( )
K, Mg, or Ca;
A is selected from NRj or O, wherein YI is selected from
cycloalkyl with up to 10 carbon atoms, linear or branched alkyl with up to 10
.""""",,
/ 1 X1
I
carbon atoms or \\X2 when A is NR', and wherein YI is selected from RI
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N
~ ' ~~ or
or CHZR when A ~s O; or AY is selected from a halogen, ,
N
and
NJ
DYz is a halogen, or D is NR' and Y2 is selected from ~' ,
~~CHR~
~NR~
or (CHR~)XNR~2, wherein x is an integer from I to 6.
Compositions comprising compounds of this formula are also
encompassed by the present invention, as well as mixtures or combinations of
compounds of this formula.
In yet another aspect of this invention, compounds of the present
invention include, but are not limited to, those having the following formula:
or an ene, a dime, a triene, or an yne derivative thereof; a saturated
derivative
thereof; a stereoisomer thereof; or a salt thereof;
wherein:
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R' is in each occurrence independently selected from -H; linear or
branched alkyl with up to 10 carbon atoms; cycloalkyl with up to 10 carbon
atO117S; aryl; or (CHZ)~CN, wherein x is an integer from 0 to 6;
E is CH or N;
n is an integer from 0 to 3;
X' is selected from -H, m-F, rn-Cl, m-Br, m-I, m-CN, m-N02, m-
SOzR', m-SOZOR', na-NC(O)R', or o-F, or X' and XZ together is a fused benzene,
pyridine, or dioxane ring;
XZ is selected from -H, o-Cl, o-Br, o-CF3, o-R', p-OR', p-SR', p-
NR'2, p-F, p-CI, p-Br, p-CF3, p-CN, p-C(O)OR', p-NC(O)R', p-(4-morpholinyl),
orp-(4-methyl-1-piperizinyl);
AY' is a halogen, or A is NR' or O and Y' is selected from
cycloalkyl with up to 10 carbon atoms, cycloalkyl with up to 10 carbon atoms
substituted with R', linear or branched alkyl with up to 10 carbon atoms,
CH2R',
~~S~CHR~
~NR~
(CHR')yOR', wherein y is an integer from 1 to 6, ; or AY' together are
N
H~C~ (CH~)x ~ wherein x is an integer from 3 to 5; and
NJ
DYZ is a halogen, or D is NR' and Ya is selected from R' ,
~~CHR~
~NR~
cycloalkyl with up to 10 carbon atoms, cycloalkyl with up to 10
carbon atoms substituted with R', linear or branched alkyl with up to 10
carbon
N
N
H2C~ \CH )X rote er from 3 to 5
atoms, CH2R , ( a , wherein x is an g , ,
CHZCF3, (CHR')ZZ', wherein z is an integer from 1 to 6, and Z' is selected
from
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_ O
O ~N~ ~N
NR~ ~ ~ HOC-(CH~)x~ wherein x is an integer from 3 to 5, ~ ,
29 7
~N~N ~ N N Z2
or ~- _~ ; or NYZR~ together is selected from ~--~ , wherein Z2
N
> > >
is selected from R , C(O)R , C(O)OR , pyndmyl, aryl, ,
(CH2)qOR~
N
O~O~ or ~ ~ , wherein q is an integer from 0 to 6.
Compositions comprising compounds of this formula are also
encompassed by the present invention, as well as mixtures or combinations of
compounds of this formula.
As an additional aspect of this invention includes compounds of
the present invention include, but are not limited to, those having the
following
formula:
R~
or an ene, a dime, a triene, or an yne derivative thereof; a saturated
derivative
thereof; a stereoisomer thereof; or a salt thereof;
wherein:
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R' is in each occurrence independently selected from -H; linear or
branched alkyl with up to 10 carbon atoms; or cycloalkyl with up to 10 carbon
atoms;
X' is selected from H, m-F, m-Cl, m-Br, m-I, m-CN, m-NO2, rn-
S02R~, or m-S020R';
XZ is selected from o-R', p-OR', p-SR', p NRIa, p-OM, or p-SM,
wherein M is selected from Li, Na, K, Mg, or Ca;
Y' is selected from cycloalkyl with up to 10 carbon atoms or
~~CHR~
~NR~
and
Y2 is selected from linear or branched alkyl with up to 10 carbon
~~CHR~
~NR~
atoms, cycloallcyl with up to 10 carbon atoms, or , and Ra is -H; or
I
N
NYZRa together is selected from HzC/ (CH~)x~ wherein x is an integer from 3 to
(CH2)qOR~
N Z2
5, ~ , wherein q is an integer from 0 to 6, or ~---~ ,
z . ~ ~N~
wherein Z ~s selected from R or .
Compositions comprising compounds of this formula are also
encompassed by the present invention, as well as mixtures or combinations of
compounds of this formula.
In still another aspect of this invention, compounds of the present
invention include, but are not limited to, those having the following
structural
formula:
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X1
2
X
R1
Y2
or an ene, a diene, a triene, or an yne derivative thereof; a saturated
derivative
thereof; a stereoisomer thereof; or a salt thereof;
wherein:
R' is in each occurrence independently selected from H; linear
or branched alkyl with up to 10 carbon atoms; or cycloalkyl with up to 10
carbon atoms;
X' is in each occurrence independently selected from -H, m-F,
m-Cl, m-Br, m-I, m-CN, m-N02, m-S02R', or m-SOZOR';
X2 is in each occurrence independently selected from o-CH3, p-
OR', p-SR', p-NR'a, orp-OM orp-SM, wherein M is selected from Li, Na, I~,
Mg, or Ca;
Y' is selected from cycloalkyl with up to 10 carbon atoms;
(CH2)n \
/ ~ X~
w\J
wherein n is I or 2; or X2 ; and
~~CHR~
NJ NR~
YZ is selected from R' or
Compositions comprising compounds of this formula are also
encompassed by the present invention, as well as mixtures or combinations of
compounds ofthis formula.
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These compounds and compositions presented above are not
intended to be limiting, but merely representative of the chemical structures
and
formulas encompassed by the present invention.
Pharmaceutically Acceptable Salts
For the proposed N2, IVY, lV6-tris(amino-substituted)-1,3,5-triazines,
the terms "non-toxic, pharmaceutically acceptable salt" or "pharmaceutically
acceptable salt" refer to a salt or complex of the 1,3,5-triazine compounds
that
retain or enhance the biologically activity of the compounds described in this
invention. Examples of salts are those that are derived from the interaction
of the
1,3,5-triazine compounds or derivatives and an inorganic (mineral acids) or
organic acid, as well as compounds derived from deprotonating an amine
nitrogen of the triamine derivatives.
Examples of inorganic acids include, but are not limited to,
hydrochloric acid, hydrobromic acid, hydroiodic acid, hydrofluoric acid,
nitric
acid, nitrous acid, perchloric acid, chloric acid, hypochlorous acid, chlorous
acid,
phosphoric acid, sulfuric acid, sulfurous acid, and carbonic acid. Examples of
organic acids include, but are not limited to acetic acid, benzene sulfonic
acid,
benzoic acid, butanoic acid, camphorsulfonic acid, citric acid, ethane
sulfonic
acid, fumaric acid, glutaric acid, 2-hydroxy acetic acid acids (derivatives
where
alkyl group is c = 3-7 and hydroxy group is located accordingly), 2-hydroxy
alkyl
sulfonic acids (derivatives where alkyl group is c = 3-7 and hydroxy group is
located accordingly), lactic acid, malefic acid, malic acid malonic, methane
sulfonic acid, naphthalene sulfonic acid, oxalic acid, palmitic acid,
propanoic
acid, phthalic acid, pyruvic acid, salicylic acid, stearic acid, succinic
acid, tartaric
acid, p-toluene sulfonic acid, and amino acids (e.g., alanine, N-
acetylglycine,
arginine, aspartic acid, glutamic acid, glycine, lysine, and phenylalanine).
Examples of salts described here include compounds that derive
from a deprotonation reaction of an amine nitrogen of the triamine derivatives
with a strong base, to form an amido salt, compound or complex. For example,
these compounds include those that are derived from the interaction or
chemical
reaction of the 1,3,5-triazine compounds or derivatives acting as a Bronsted
or
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Lewis acid and an inorganic or organic base to form an ionic and/or complexed
species Examples of inorganic bases, include but not limited to, metallic
bases or
organometallic bases such as alkyllithiums or metal hydrides, where there is a
metallic counterion include, but are not limited to, aluminum, barium,
calcium,
lithium, magnesium, potassium sodium, and zinc.
Examples of organic bases include, but are not limited to, alky and
aryl amines as well as ammonia. Included in this description are salts formed
from the combination or interaction/reaction of inorganic acids (e.g., Lewis
acids) and metallic counterions and the 1,3,4-triazine compounds or
derivatives
acting as a Bronsted or Lewis base resulting in the formation of ionic and/or
complexed species For all salts and complexes as described above, these are to
include hydrated or solvated forms of the compounds.
Additionally, this invention also encompasses salts of these
triazine derivatives that are non-toxic and pharmaceutically acceptable, such
as
quaternary ammonium salts, for example [-N+RZR~]X-, where the R and R groups
represent hydrogen or an organic group (such as alkyl, alkenyl, alkynyl, aryl,
and
the like) and the X group is a counter ion (halogen, hydroxide, alkoxide,
thioalkoxide, or conjugate base of an organic or inorganic acid). For all
salts and
complexes as described above, these are to include hydrated or solvated forms
of
the compounds.
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III. ANTIPROLIFERATIVE ACTIVITIES
One embodiment of the present invention comprises methods and
compositions comprising the compounds of the present invention for the
treatment and prevention of conditions or diseases that have as an aspect of
the
disease or condition, unwanted cellular proliferation occurring or are the
result of
cellular proliferation. For example, many vascular diseases, such as
cardiovascular diseases, organ transplant sequellae, vascular occlusive
conditions
including, but not limited to, neointimal hyperplasia, restenosis, transplant
vasculopathy, cardiac allograft vasculopathy, atherosclerosis, and
arteriosclerosis.
are caused by or have collateral damage due to unwanted cellular
proliferation.
Smooth muscle cell (SMC) hyperplasia is a major event in the development of
atherosclerosis and is also responsible for the significant number of failure
rates
following vascular procedures such as angioplasty and coronary artery bypass
surgery, particularly due to restenosis. Proliferation of arterial wall SMC in
response to local injury is a major feature of many vascular proliferative
disorders. Neointimal hyperplasia is commonly seen after various forms of
vascular injury and a major component of the vein graft's response to harvest
and
surgical implantation into high-pressure arterial circulation. Proliferation
of
SMC in response to local injury is a major feature of vascular proliferative
disorders such as atherosclerosis and restenosis after angioplasty.
One aspect of the present invention relates to methods and compositions
for the treatment and prevention of smooth muscle cell (SMC) proliferation,
preferably comprising compositions and compounds having cellular
antiproliferative activity. These compounds and compositions comprising such
compounds are referred to as antiproliferative compounds or compositions. At
least one activity of one or more of these compounds is that the compound has
the activity of effecting the synthesis of proteoglycans including induction
and
synthesis of proteoglycans and active fragments of proteoglycans. Thus, one
aspect of the activity of one or more of the compounds and compositions of the
present invention comprise molecules that induce HSPG production and that
regulate SMC (smooth muscle cell) proliferation.
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Compounds of the present invention that have at least the activity of
effecting cellular proliferation are shown in TABLE 3. The compounds shown in
this Table have the activity of effecting cellular proliferation as measured
by the
assays taught herein. The inclusion of compounds in the categories of the
Tables
disclosed herein are not to be seen as limiting, in that compounds included in
such Tables have at least the activity shown for inclusion in the Table and
may
have more or other activities. Nor are the Tables to be seen as limiting in
that
these are the only compounds disclosed herein that have that activity,
representative compounds are shown in the Tables that have at least that
particular activity for inclusion in the Table. One or more compounds
disclosed
herein have at least an activity that has utility in treatment of disease
states.
Examples of compounds that show at least this activity and utility are
shown in the following structure:
R
or an ene, a dime, a triene, or an yne derivative thereof; a saturated
derivative
thereof; a stereoisomer thereof; or a salt thereof;
wherein:
R~ is in each occurrence independently selected from -H; linear or
branched alleyl with up to 10 carbon atoms; or cycloalkyl with up to 10 carbon
atoms;
X' is selected from rn-F, m-Cl, m-Br, m-I, m-CN, m-N02, m-S02R1, or m-
SOZOR~, or X~ and XZ together is a fused benzene, pyridine, or dioxane ring;
Xa is selected from p-OR', p-SR', p-NR12, p-OM, or p-SM, wherein M is
selected from Li, Na, K, Mg, or Ca;
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Y' is selected from cycloalkyl with up to 10 carbon atoms; linear or
branched alkyl with up to 10 carbon atoms; CHaR2, wherein R2 is a cycloalkyl
(CH2)n
N
with up to 10 carbon atoms; or ~ , wherein n is 1 or 2;
AYz is selected from a halogen or OR', or
~~CHR~
NR~
N
A is NR' and YZ is selected from R', R~ , or
Further examples of compounds that show at least this activity and utility are
presented in Table 3, where compound activity is also presented. The activity
scale used in Table 3 as follows (numbers are inclusive): "+-!-I-" represents
ICso
of less than about 3 p.M; "++" represents ICso of between about 3 and about 7
pM; and "+" represents ICso of greater than about 7 p,M. Further, any hydrogen
atoms that are required for any atom to attain its usual valence in a
structure
presented in Table 3, whether a carbon atom or a heteroatom, should be
inferred
if it is not specifically indicated.
In addition to the above compounds, the following compounds and
compositions comprising these compounds are active in an anti-proliferation
assay (Perlecan). These compounds and compositions comprising these
compounds are, among other things, generally useful for treating
cardiovascular
disorders associated with proliferative activity. Specifically, these
compounds
include Na-cycloheptyl 1V4-(3-fluoro-4-methoxyphenyl)-1V6-methyllV6-(1-methyl-
piperidin-4-yl)-1,3,5-triazine-2,4,6-triamine, and N2-cycloheptyl-N4-methyl N4-
( I -methyl-piperidin-4-yl)-1Vg-naphthalen-2-yl-1,3,5-triazine-2,4,6-triamine.
Using the same activity scale used in Table 3, and discussed above, the first
compound, Na-cycloheptyl-1V4-(3-fluoro-4-methoxyphenyl)-1V6-methy11V6-(1
methyl-piperidin-4-yl)-1,3,5-triazine-2,4,6-triamine, is characterized as a
compound exhibiting medium or moderate activity, while the second compound,
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NZ-cycloheptyl-lV4-methyl-lV4-( I -methyl-piperidin-4-yl)-1V6-naphthalen-2-yl-
1,3,5-triazine-2,4,6-triamine, is characterized as a compound exhibiting high
activity.
As used herein, when a proteoglycan is referred to, the entire molecule or
fragments are included therein. For example, perlecan refers to the entire
perlecan molecule or fragments thereof. Different fragments of perlecan may
have the same or different effects on cells and the effects may be the same as
or
different from the effects that the entire perlecan molecule has on cells.
These
fragments and activities are contemplated in the present invention and
compounds included in the present invention may have at least one activity
that
modulates or effects the fragements' activities or the entire molecule's
activities.
Although the discussion herein refers specifically to perlecans it is
important to
note that the compositions, methods, and assays described herein are equally
applicable in the context of other proteoglycans, including HSPGs, and
including
but not limited to, chondroitan sulfates (e.g., A,B, and C), dermatan
sulfates,
syndecans and glypicans.
Methods for identifying the activity and screening for one or more of
these compounds or molecules that induce synthesis of proteoglycans such as
HSPG (heparan sulfate proteoglycan) are taught in U.S. Patent Application No.
10/091,357, which is incorporated herein in its entirety. Assays of effects of
compounds in vivo are also taught in the incorporated references and are known
to those skilled in the art. In general, methods comprise the addition of such
compounds to assays and measurement of HSPG synthesis including, but not
limited to, the production of syndecans, glypicans and perlecans, for example,
syndecans 1, 2 and 4; and glypican-1. Other assays that can be used to
determine
the activity of the compounds of the present invention include other methods
for
measuring the induction of perlecan synthesis. For example, in one assay,
perlecan is induced in cells by certain inducers, and the response is
measured.
Compounds of the present invention are then added to a replicate assay and the
effect on perlecan induction is determined. Using such methods, compounds are
determined that can either inhibit perlecan, elevate induction of perlecan, or
that
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have no effect at all. Those compounds that are effective as therapeutic
agents
can then be used in animals, humans or patients with cellular proliferation
disease
aspects, such as vascular-associated diseases or SMC proliferation
pathologies.
Another assay for determining compounds having SMC effects comprises
adding a composition suspected of effecting SMC proliferation to smooth muscle
cells in growth medium or serum-free medium. The change in cell proliferation
can be measured by methods known to those skilled in the art, such as
incorporation of labeled nucleotides into dividing cells' DNA, and compared to
the proliferation of cells which are not treated with the compound. Other
measurements include directly determining levels of HSPG synthesis by
measuring the amount or change in amount of HSPG such as with ELISA for
HSPGs, and compared to the amount of HSPG synthesis in untreated cells. Other
indirect or direct measurement are contemplated by the present invention and
are
known to those skilled in the art. For example, such methods include, but are
not
limited to, measurement of RNA levels, RT-PCR, Northern blotting, Western
blotting promoter-based assays to identify compounds that affect one or more
proteoglycans and assays for proteoglycan biological activity shown by
recombinant proteins, partially purified proteins, or lysates from cells
expressing
proteoglycans in the presence or absence of compounds of interest.
An assay for identifying and determining an activity of one or more of the
compounds of the present invention comprises identifying compounds that
interact with the promoter regions of a gene, or interact and effect proteins
that
interact with the promoter region, and are important in the transcriptional
regulation of the protein's expression. For example, if perlecan were the
protein,
in general, the method comprises a vector comprising regulatory sequences of
the
perlecan gene and an indicator region controlled by the regulatory sequences,
such as an enzyme, in a promoter-reporter construct. The protein product of
the
indicator region is referred to herein as a reporter enzyme or reporter
protein.
The regulatory region of the sequence of perlecan comprises a range of
nucleotides from approximately -4000 to +2000 wherein the transcription
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initiation site is +l, more preferably, from -2500 to +1200, most preferably,
from
-I 500 to +800 relative to the transcription initiation site.
Cells are transfected with a vector comprising the promoter-reporter
construct and then treated with one or more compositions comprising at least
one
compound of the present invention. For example, the transfected cells are
treated
with a composition comprising a compound suspected of effecting the
transcription of perlecan and the level of activity of the perlecan regulatory
sequences are compared to the level of activity in cells that were not treated
with
the compound. The level of activity of the perlecan regulatory sequences are
determined by measuring the amount of the reporter protein or determining the
activity of the reporter enzyme controlled by the regulatory sequences. An
increase in the amount of the reporter protein or the reporter enzyme activity
shows a stimulatory effect on perlecan, by positively effecting the promoter,
whereas a decrease in the amount or the reporter protein or the reporter
enzyme
activity shows a negative effect on the promoter and thus, on perlecan.
Additionally, the present invention comprises methods and compositions
that can be used with gene therapy methods and composition, such as those gene
therapy methods comprising administering compositions comprising nucleic
acids that effect the synthesis or expression of HSPGs, particularly perlecan.
Such methods and compositions are taught in U.S. Patent Application No.
10/091,357, incorporated herein by reference.l
The present invention comprises methods and compositions for mediating
proteoglycan synthesis, expression and for the maintenance of SMC in a
quiescent state. Methods and compositions of the present invention comprise
~ RICK- How does this apply to this invention- compounds that treat disease?
There is no discussion or support for transfection of genes to affect HSPG
synthesis. Should we cut this paragraph and leave it to the application cited?
(18631-0141 ).
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treatment and prevention of vascular diseases and pathologies related to
celluar
proliferation, such as SMC proliferation. Such methods and compositions
comprise methods for inhibition of smooth muscle cell (SMC) growth and
proliferation, and for induction of quiescence in smooth muscle cells.
Embodiments of the present invention comprise methods and compositions for
inducing proteoglycan synthesis, particularly HSPG synthesis and expression
including, but not limited to, the induction of HSPGs such as syndecans,
glypicans and perlecans, and preferably perlecan synthesis and gene
expression.
Perlecan is a major extracellular HSPG in the blood vessel matrix. It
interacts
with extracellular matrix proteins, growth factors and receptors. Perlecan is
also
present in basement membranes other than blood vessels and in other
extracellular matrix structures.
The activities of the compounds included in the present invention effect
cells or tissues to increase the synthesis of proteoglycans by those cells or
tissues
or may act directly upon one or more proteoglycans to modulate the biological
activity or to increase the biological stability of the proteoglycan itself,
for
example, of the protein perlecan. Activities also included herein are ones
that
increase the biosynthesis of one or more proteoglycans by increasing the
transcription of the poteoglycan gene, increasing the biological stability of
the
proteoglycan mRNA or increasing the translation of proteoglycan mRNA into
protein. Further activites include activities of compounds that can block or
decrease the effects of agents or proteins that inhibit the activity of
proteoglycans.
The present invention comprises methods and compositions for the
treatment and prevention of smooth muscle cell proliferation, including
vascular
occlusive pathologies. Such methods comprise administration of compositions
comprising compounds capable of inhibiting SMC proliferation, such as
compositions comprising compounds disclosed herein that inhibit SMC
proliferation. Administration of such compounds that are effective in
inhibiting
SMC proliferation are administered to humans and animals suspected of having
or who have, for example, vasculopathy or who have undergone angioplasty or
other procedures damaging to the endothelium. Effective amounts are
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administered to such humans and animals in dosages that are safe and
effective,
including, but not limited to, the ranges taught herein. Routes of
administration
include, but are not limited to, those disclosed herein. As disclosed herein,
compositions comprising such compounds may be used in conjunction with other
therapeutic agents or in methods comprising steps such as altered patient
activities, including, but not limited to, changes in exercise or diet.
The compounds of the present invention are useful in the treatment or
prophylaxis of at least one cardiovascular disease in a cell, tissue, organ,
animal,
or patient including, but not limited to, vascular occlusive lesions including
atherosclerosis, transplant vasculopathy, cardiac allograft vasculopathy,
restenosis, graft atherosclerosis after coronary transplantation, cardiac stun
syndrome, myocardial infarction, congestive heart failure, stroke, ischemic
stroke, hemorrhage, arteriosclerosis, atherosclerosis, restenosis, diabetic
ateriosclerotic disease, hypertension, arterial hypertension, renovascular
hypertension, syncope, shock, syphilis of the cardiovascular system, heart
failure,
cor puhnonale, primary pulmonary hypertension, cardiac arrhythmias, atrial
ectopic beats, atrial flutter, atrial fibrillation (sustained or paroxysmal),
post
perfusion syndrome, cardiopulmonary bypass inflammation response, chaotic or
multifocal atrial tachycardia, regular narrow QRS tachycardia, specific
arrythmias, ventricular fibrillation, His bundle arrythmias, atrioventricular
block,
bundle branch block, myocardial ischemic disorders, coronary artery disease,
angina pectoris, myocardial infarction, cardiomyopathy, dilated congestive
cardiomyopathy, restrictive cardiomyopathy, valvular heart diseases,
endocarditis, pericardial disease, cardiac tumors, aordic and peripheral
aneuryisms, aortic dissection, inflammation of the aorta, occulsion of the
abdominal aorta and its branches, peripheral vascular disorders, occulsive
arterial
disorders, peripheral atherlosclerotic disease, thromboangitis obliterans,
functional peripheral arterial disorders, Raynaud's phenomenon and disease,
acrocyanosis, erythromelalgia, venous diseases, venous thrombosis, varicose
veins, arteriovenous fistula, lymphederma, lipedema, unstable angina,
reperfusion
injury, post pump syndrome, ischemia-reperfusion injury, and the like. Such
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methods can optionally comprise administering an effective amount of a
composition or pharmaceutical composition comprising at least one compound to
a cell, tissue, organ, animal or patient in need of such modulation, treatment
or
therapy.
Proteoglycan-associated diseases that are treatable with the compounds of
the present invention include, but are not limited to, hereditary multiple
exostosis,
mucopolysaccharidosis types I-III and VII, commonly known as Hurler's
Syndrome, Hunter's Syndrome, Sanfilippo's Syndrome and Sly's Syndrome
respectively, Alzheimer's disease, Simpson-Golabi-Behmel syndrome, fibroblast
growth factor related disorders, herpes simplex virus, dengue fever,
Parkinson's
disease, renal disease, muscular dystrophy, Schwarts-Jampel syndrome,
proteinuric glomerulopathies, myotonia and skeletal dysplasia, kyphoscoliosis,
dyssegmental dysplasia, Silverman-Handmaker type, chondrodysplasia,
periodontitis, rheumatoid and osteoarthritis, Gerstmann-Straussler syndrome,
Creutzfeldt-Jakob disease, scrapie, carcinomas, Happle syndrome, macular
dystrophy, bone diseases, corneal diseases, leukocyte-mediated disease,
collagen
fibril assembly disorder and coronary heart disease and other vascular
disorders.
IV. Glycosidase Modulation Activity
The present invention also comprises methods and compositions
comprising compounds described herein that have an activity associated with
modulation of glycosidase enzymes and thus, effecting the substrates for such
enzymes. Glycosidase enzymes and their activity with their substrates, such as
proteoglycans or glycated proteins, are aspects of a variety of diseases such
as
vascular conditions, including those conditions discussed supra, proteoglycan-
associated diseases, supra, associated diseases with vascular components,
including but not limited to, kidney disease, ischemic heart disease,
cardiovascular disease, generalized vascular disease, proliferative
retinopathy,
and macroangeopathy, inflammatory diseases and metastatic diseases such as
cancer, cellular proliferative conditions, and solid and blood borne tumors or
other oncological conditions. Compounds described herein that have an activity
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that effects the concentrations of substrates of glycosidase enzymes are used
in
methods of treatment of such vascular, inflammatory, metastatic and systemic
diseases.
An aspect of the present invention comprises methods and compositions
for the modulation of enzymes, such as glycosaminoglycan degrading enzymes,
which effect or are effected by proteoglycan levels, amount or activity. For
example, the present invention comprises methods and compositions comprising
compounds that modulate enzymes including but not limited to, heparanase,
chondroitanase, heparan sulfate endoglycosidase, heparan sulfate
exoglycosidase,
polysaccharide lyases, keratinase, hyauronidase, glucanase, amylase,
glycosidases, or other proteoglycan degrading enzymes are useful for the
treatment of conditions such as diabetic vasculopathy, cancer, inflammatory
diseases, autoimmune diseases and cardiovascular diseases. For example, the
present invention comprises methods and compositions of compounds that
I S inhibit, impair or down-regulate the activity of proteoglycan degrading
enzymes.
Proteoglycans such as HSPG are important components of the
subendothelial extracellular matrix and the basement membrane of blood
vessels.
Rosenberg et al., 99 J. CLIN. INVEST. 2062-70 (1997). Basement membranes are
continuous sheets of extracellular matrix composed of collagenous and
noncollagenous proteins and proteoglycans that separate parenchyma) cells from
underlying interstitial connective tissue. They have characteristic
permeabilities
and play a role in maintaining tissue architecture.
In addition to HSPGs, the basal lamina consists predominantly of a
complex network of adhesion proteins, fibronectin, laminin, collagen and
vitronectin. Wight et al., 6 CURB. QPIN. LIPIDOL. 326-334 (1995). Heparan
sulfate (HS) is an important structural component of the basal lamina. Each of
the adhesion proteins interacts with HS side chains of HSPGs within the
matrix.
Thus, HSPGs function as a barrier to the extravasation of metastatic and
inflammatory cells. Cleavage of HS by the endoglycosidase heparanase
produced by metastatic tumor cells and inflammatory cells destroys the
filtering
properties of the lamina. In addition, the degradation of the HS may assist in
the
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disassembly of the extracellular matrix and thereby facilitate cell migration
by
allowing blood borne cells to escape into the bloodstream. Vlodavsky et al.,
12
INVASION METASTASIS I 12-127 (1992).
Heparanase activity has been described in a number of tissues and cell
types including liver, placenta, platelets, fibroblasts, neutrophils,
activated T and
B-lymphocytes, monocytes, and endothelial cells (7-16). Nakajima et al., (31)
CANCER LETT. 277-283 (1986); Nakajima et al., 36 J. CELL. BIOCHEM. 157-167
(1988); Ricoveri et al., 46 CANeER RES. 3855-3861 (1986); Gallagher et al.,
250
BIOCHEM. J. 719-726 (1988); Dempsey et al., 10 GLYCOBIOLQGY 467 (2000);
Goshen et al., 2 Mol.. HUM. REPROD. 679 (1996); Parish et al., 76 IMMUNOL
CELL BIOL. 104-I 13 (1998); Gilat et a(, 181 J. EXP. MED. 1929-1934 (1995);
Graham, et al., 39 BIOCHEM. Mol.. BIOL. INT. 56371 (1996); Pillarisetti et
al.,
270 J.BIOL.CHEM. 29760-29765 (1995). An important process in tissue invasion
by blood-borne tumor cells and white cells involves their passage through the
vascular endothelial cell layer and subsequent degradation of the underlying
basal lamina or basement membranes and extracellular matrix with a battery of
secreted proteases and glycosidases. Nakajinia et al., 220 SCIENCE 611-613
(1983); Vlodavsky et a1.,12 INVASION METASTASIS 112-127 (1992).
Heparanase activity was shown to correlate with the metastatic potential
of animal and human tumor cell lines. Nakajima et al., 31 CANCER LETT. 277-
283 (1986); Nakajima et al., 212 PROD CLIN BIOL RES. 113-122 (1986); Freeman
et al., 325 BIOCNEM. J. 229-237 (1997); Vlodavsky et al., S NAT. MED. 793-802
(1999); Hulett et al., S NAT MED. 803-809 (1999). It is also known to regulate
growth. factor activity. Many growth factors remain bound to heparan sulfate
in
storage form and are disassociated by heparanase during angiogenesis,
improving
the survival rate of cancer cells.
Serum heparanase levels in rats were higher by more than an order of
magnitude after injection of the rats with highly metastatic mammary
adenocarcinoma cells. In addition, heparanase activity in the sera of rats
bearing
MTLn3 tumors correlated well with the extent of the metastases. Moreover,
serumlurine heparanase activity in cancer patients was shown to be 2-4 fold
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increased in particular where tissue metastases were present. Because the
cleavage of HS appears to be essential for the passage of metastatic tumor
cells
and leukocytes through basement membranes, studies of heparanase inhibitors
provides the potential of developing a novel and highly selective class of
anti
s metastatic and anti-inflammatory drugs.
The present invention comprises methods and compositions comprising
compounds that modulate heparanase activity or the activity of other
glycosidases, including, but not limited to enzymes with glycosaminoglycan
activity such as chondroitinase, heparan sulfate endoglycosidase, heparan
sulfate
exoglycosidase, polysaccharide lyases, leeratinase, hyaluranidase, glucanase,
and
amylase. Compounds of the present invention that have at least the activity of
modulating glycosidase enzyme activity are shown in TABLE 6. The
compounds shown in this Table have the activity of modulating glycosidase
enzyme activity as measured by the assays taught herein. The inclusion of
compounds in the categories of the Tables disclosed herein are not to be seen
as
limiting, in that compounds included in such Tables have at least the activity
shown for inclusion in the Table and may have more or other activities. Nor
are
the Tables to be seen as limiting in that these are the only compounds
disclosed
herein that have that activity, representative compounds are shown in the
Tables
that have at least that particular activity for inclusion in the Table. One or
more
compounds disclosed herein have at least an activity that has utility in
treatment
of disease states.
Examples of compounds that show at least this activity and utility are
shown in the following formula:
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R
X1
R1
~N
X2
N~
1 ~ 2
R
\11 ~ 12
Y Y
or an ene, a dime, a triene, or an yne derivative thereof; a saturated
derivative
thereof; a stereoisomer thereof; or a salt thereof;
wherein:
R' is in each occurrence independently selected from H; linear or
branched alkyl with up to 10 carbon atoms; or cycloalkyl with up to 10 carbon
atoms;
X' is selected from H, m-F, m-Cl, m-Br, m-I, m-CN, m-NO2, m-SOaR~, or
m-SOZOR~;
X2 is selected from o-R~, p-ORS, p-SRS, p-NR~2, p-OM, orp-SM, wherein
M is selected from Li, Na, I~, Mg, or Ca;
.~~CHR~
~NR~
Y' is selected from cycloalkyl with up to 10 carbon atoms or ;
and
Y2 is selected from linear or branched alkyl with up to 10 carbon atoms,
~HR~
~NR~
cycloalkyl with up to 10 carbon atoms, or , and Ra is -H; or NYaR2
N
together is selected from H~C~ (CH2)x, wherein x is an integer from 3 to 5,
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~~CH~)qOR~
N~ Z2
wherein q is an integer from 0 to 6, or
O
N
wherein Z2 is selected from R' or ~ .
Further examples of compounds that show at least this activity and utility
are presented in Table 6, where compound activity is also shown. The activity
scale used in Table 6 is as follows (numbers are inclusive): "-+++" represents
between about 70 and about 100% inhibition; "-+-E-" represents between about
30
and about 40% inhibition; and "+" indicates between 0 and about 30%
inhibition,
all at 5 pM compound concentration. Also note that any hydrogen atoms that are
required for any atom to attain its usual valence in a structure presented in
Table
6, whether a carbon atom or a heteroatom, should be inferred if it is not
specifcally indicated.
Compounds or compositions comprising such compounds that are
effective in modulating glycosidase enzyme activity are useful in treating
and/or
preventing cancer including, but not limited to, malignant and non-malignant
cell
growth, leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), B-cell,
T-cell or FAB ALL, acute myeloid leukemia (AML), chromic myelocytic
leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia,
myelodyplastic syndrome (MDS), a lymphoma, Hodgkin's disease, a malignamt
lymphoma, non-hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma,
Kaposi's sarcoma, colorectal carcinoma, pancreatic carcinoma, nasopharyngeal
carcinoma, malignant histiocytosis, paraneoplastic syndrome/hypercalcemia of
malignancy, solid tumors, adenocarcinomas, sarcomas, malignant melanoma,
hemangioma, metastatic disease, cancer related bone resorption, cancer related
bone pain, and the like.
1n another aspect of the present invention, the compounds disclosed
herein are useful in modulating heparanase activity or the activity of other
glycosidases as a means for treating and preventing autoimmune diseases.
Generally autoimmune disease results when (I) the immune system mistakenly
identifies a cell surface molecule on normal tissue as a foreign molecule (2)
the
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synthesis and secretion of chemokines, cytokines and lymphokines is not shut
down after the eradication of the disease or (3) the immune system overreacts
to
the apparent infection and destroys vast quantities of surrounding normal
tissue.
To be effective in an immune response, the immune effector cells must
S bind to the luminal/apical surface of the blood vessel walls. This is
accomplished
through the interaction of adhesion molecules on the immune effector cells
with
their locally upregulated cognate receptors on the endothelial cells lining
the
vasculature near the site of infection. After binding to the apical surface
and
before entering the inflamed tissue, the immune effector cells must breach the
basement membrane (BM) and extracellular matrix (ECM) that surround the
basal portion of the blood vessels and give the vessels their shape and
strength.
The BM and ECM consists of structural proteins embedded in a fiber meshwork
consisting mainly of complex carbohydrate containing structures
(glycosaminoglycans), of which the main constituent is heparin sulfate
I S proteoglycan (HSPG). In order to breach this barrier the immune effector
cell
must weaken or destroy it, which is accomplished through the local secretion
of
proteases and heparanase(s).
Thus, the inhibition of heparanase or the activity of other glycosidases
using the compounds of the present invention finds utitlity in treating
arthritis and
other autoimmune diseases. More specifically, the compounds of the present
invention are useful in the treatment or prophylaxis of at least one
autoimmune-
related disease in a cell, tissue, organ, animal, or patient including, but
not limited
to, rheumatoid arthritis, juvenile rheumatoid arthritis, systemic onset
juvenile
rheumatoid arthritis, psoriatic arthritis, ankylosing spondilitis, gastric
ulcer,
seronegative arthropathies, osteoarthritis, inflammatory bowel disease,
ulcerative
colitis, systemic lupus erythematosis, antiphospholipid syndrome,
iridocyclitis/uveitis/optic neuritis idiopathic pulmonary fibrosis, systemic
vasculitis/wegener's granulomatosis, sarcoidosis, orchitis/vasectomy reversal
procedures, allergic/atopic diseases, asthma, allergic rhinitis, eczema,
allergic
contact dermatitis, allergic conjunctivitis, hypersensitivity pneumonitis,
transplants, organ transplant rejection, graft-versus-host disease, systemic
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inflammatory response syndrome, sepsis syndrome, gram positive sepsis, gram
negative sepsis, culture negative sepsis, fungal sepsis, neutropenic fever,
urosepsis, meningococcemia, traumalhemorrhage, burns, ionizing radiation
exposure, acute pancreatitis, adult respiratory distress syndrome, rheumatoid
arthritis, alcohol-induced hepatitis, chronic inflammatory pathologies,
Crohn's
pathology, sickle cell anemia, diabetes, nephrosis, atopic diseases,
hypersensitity
reactions, allergic rhinitis, hay fever, perennial rhinitis, conjunctivitis,
endometriosis, asthma, urticaria, systemic anaphalaxis, dermatitis, pernicious
anemia, hemolytic disesease, thrombocytopenia, graft rejection of any organ or
tissue, leidney transplant rejection, heart transplant rejection, liver
transplant
rejection, pancreas transplant rejection, lung transplant rejection, bone
marrow
transplant (BMT) rejection, skin allograft rejection, cartilage transplant
rejection,
bone graft rejection, small bowel transplant rejection, fetal thymus implant
rejection, parathyroid transplant rejection, xenograft rejection of any organ
or
I S tissue, allograft rejection, anti-receptor hypersensitivity reactions,
Graves disease,
Raynoud's disease, type B insulin-resistant diabetes, asthma, myasthenia
gravis, -
meditated cytotoxicity, type III hypersensitivity reactions, POEMS syndrome
(polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and
skin changes syndrome), polyneuropathy, organomegaly, endocrinopathy,
monoclonal gammopathy, skin changes syndrome, anti-phospholipid syndrome,
pemphigus, scleroderma, mixed connective tissue disease, idiopathic Addison's
disease, autoimmune hemolytic anemia, autoimmune hepatitis, idiopathic
pulmonary fibrosis, scleroderma, diabetes mellitus, chronic active hepatitis,
vitiligo, vasculitis, post-MI cardiotomy syndrome, type IV hypersensitivity ,
contact dermatitis, hypersensitivity pneumonitis, allograft rejection,
granulomas
due to intracellular organisms, drug sensitivity, metabolic/idiopathic,
Wilson's
disease, hemachromatosis, alpha-I-antitrypsin deficiency, diabetic
retinopathy,
hashimoto's thyroiditis, osteoporosis, hypothalamic-pituitary-adrenal axis
evaluation, primary biliary cirrhosis, thyroiditis, encephalomyelitis,
cachexia,
cystic fibrosis, neonatal chronic lung disease, chronic obstructive pulmonary
disease (COPD), familial hematophagocytic lymphohistiocytosis, dermatologic
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conditions, psoriasis, alopecia, nephrotic syndrome, nephritis, glomerular
nephritis, acute renal failure, hemodialysis, uremia, toxicity, preeclampsia,
anleylosing spondylitis, Behcet's disease, bullous pemphigoid, cardiomyopathy,
celiac spree-dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS),
chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome,
cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, discoid
lupus,
essential mixed cryoglobulinemia, flbromyalgia-fibromyositis, Graves' disease,
Guillain-Barre, Hashimoto's thyroiditis, idiopathic thrombocytopenia purpura
(ITP), IgA nephropathy, insulin dependent diabetes, juvenile arthritis, lichen
planes, meniere's disease, multiple sclerosis, pemphigus vulgaris,
polyarteritis
. nodosa, Cogan's syndrome, polychondritis, polyglandular syndromes,
polymyalgia rheumatica, polymyositis and dermatomyositis, primary
agammaglobulinemia, Raynaud's phenomenon, Reiter's syndrome, rheumatic
fever, Sjogren's syndrome, stiff man syndrome, Takayasu arteritis, temporal
I S arteritis/giant cell arteritis, Wegener's granulomatosis; okt3 therapy,
anti-cd3
therapy, cytokine therapy, chemotherapy, radiation therapy (e.g., including
but
not limited toasthenia, anemia, cachexia, and the like), chronic salicylate
intoxication, and.the like.
Compounds having heparanase activity inhibition, that are effective for
example, in treatment of cancer and autoimmune disease, can be determined
using assays such as those disclosed in U.S. Patent Application No.
09/952,648,
which is incorporated herein in its entirety. Such assays, which are used for
measurement of cellular and enzymatic activities, both qualitatively and
quantitatively, and in methods for diagnosing metastases, metastatic potential
and
inflammatory states, are performed with and without the addition of at least
one
of the compounds of the present invention to determine the activity of the
compound. Existing heparanase assays are taught in Goshen et al., 2 MoL. HuM.
REPROD. 679-84 (1996); Nakajima et al., 31 CANCER LETr. 277-83 (1986); and
Vlodasky et al., 12 INVASION METASTASIS 112-27 (1992); Freeman and Parish,
325 BtocHEtvt. J. 229-37 (1997); Kahn and Newman, 196 ANAL. BIOCHEM. 373-
76 (1991).. Solid-phase heparanase assays have also been developed where
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chemically and biosynthetically radiolabeled heparin and HS chains were
attached to a solid support, with release of radiolabel from the solid support
being
a measure of enzyme activity. Assays using such procedures are taught in U. S.
Patent No. 4,859,581, which is entirely expressly herein incorporated by
reference.
In general, a preferred assay comprises attaching one of a binding partner
to a substrate for the enzyme to be measured, forming the substrate-binding
partner. Incubation with a sample comprising the enzyme to be measured allows
for activity by the enzyme to be measured in a reaction mixture. A portion or
the
whole reaction mixture, depending on the amount needed, is then mixed with the
complementary binding partner, so that the binding partners are bound
together.
This is the first binding reaction. After incubating to allow for binding,
washings
are performed. A complementary binding partner, complementary to the first
binding partner attached to the substrate, is added. This complementary
binding
partner may or may not be the same as the first complementary binding partner.
This is the second binding reaction. The complementary binding partner in the
second binding reaction is labeled in a manner that is detectable. For
example,
the complementary binding partner is labeled with an enzyme that causes a
detectable color change when the appropriate reaction conditions exist. The
difference between the activity of the enzyme in the presence of a compound
and
the absence of compound is used to determine the activity of the compound.
An example of a heparanase assay comprises the following steps. A
composition comprising biotin-HS (heparan sulfate) is mixed with a biological
sample such as a tumor sample, bodily fluid, or other fluid suspected of
having
heparanase activity, to form a reaction mixture. This sample may be pretreated
to
remove contaminating or reactive substances such as endogenous biotin. A
control portion for this reaction mixture does not contain a compound of the
present invention, whereas a test portion contains one or more compounds
disclosed herein. After incubation, an aliquot or portion of the reaction
mixture
portions is removed and placed in a biotin-binding plate. The biotin-binding
plate comprises any means for binding biotin, preferably to a solid surface.
See
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WO 02/23197, which is entirely expressly incorporated herein by reference.
After washing with buffers, a streptavidin-enzyme conjugate is added to the
biotin-binding plate. Reagents for the enzyme are added to form a detectable
color product. For example, a decrease in color formation, from a known
standard, indicates there was heparanase activity in the sample. The
difference
between the activity of the enzyme in the presence of a compound and the
absence of compound is used to determine the activity of the compound.
Using the above assays or those taught in the Examples herein, the
amount of enzyme activity in a sample can be determined and the activities of
compounds of the present invention can be determined. For example, a
composition comprising a compound of the present invention is added to a
lenown amount of heparanase either before or during the incubation of the
heparanase and its substrate-binding partner. If the compound alters the
activity
of the heparanase, the assay methods of the present invention will show a
change
in the amount of detectable label. Such assays are used for high throughput
determination of the activity of compounds. See WO 02/23197, which is entirely
expressly incorporated herein by reference.
The activities of the compounds included in the present invention
modulate the activity of glycosidases, either positively or negatively,
include
effects on the glycosidases either directly or indirectly. The compounds may
modulate the synthesis of glycosidases by cells or tissues or may act directly
upon one or more glycosidases to modulate the biological activity or the
biological stability of the enzyme itself, for example, heparanase. Activities
also
included herein are ones that increase the biosynthesis, of one or more
glycosidases by increasing the transcription of the glycosidase gene,
increasing
the biological stability of the glycosidase mRNA or increasing the translation
of
glycosidase mRNA into protein. Further activites include activities of
compounds that can block or decrease the effects of agents or proteins that
inhibit
the activity of glycosidases. Additionally, acitivities are included that
effect the
substrates for the glycosidases, such as those discussed supra in relation to
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proteoglycans, or effect the binding parameters of the enzyme with its
substrate,
cofactors or stimulatory or inhibitory factors.
The present invention comprises methods and compositions for the
treatment and prevention of diseases or conditions that present or result from
glycosidase activity. Such methods comprise administration of compositions
comprising compounds capable of modulating heparanase activity, such as
compositions comprising compounds disclosed herein that inhibit heparanase
activity. Administration of such compounds that are effective in modulating
heparanase activity are administered to humans and animals suspected of having
or who have, for example, inflammatory conditions, autoimmune disease or
diabetic vasculopathy. Effective amounts are administered to such humans and
animals in dosages that are safe and effective, including, but not limited to,
the
ranges taught herein. Routes of administration include, but are not limited
to,
those disclosed herein. As disclosed herein, compositions comprising such
I S compounds may be used in conjunction with other therapeutic agents or in
methods comprising steps such as altered patient activities.
V. Inflammation Modulation
An embodiment of the present invention comprises methods and
compositions comprising compounds of the present invention for the treatment
and prevention of conditions or diseases that have as an aspect of the disease
or
condition, inflammation. An aspect of the present invention is directed to
methods and compositions comprising compounds that are effective in inhibiting
inflammation, particularly inflammation associated with the accumulation or
presence of glycated proteins or AGE. The activity of modulating inflammation
includes, but is not limited to, inhibiting inflammation and/or its associated
cell
activation by glycated proteins or AGE, blocking the glycation of proteins,
blocking AGE interactions with receptors, blocking AGE-induced signaling or
signaling-associated inflammatory responses, cytokine induction, synthesis or
release, AGE formation or AGE cross-linking.
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The present invention also provides compositions for and methods of
treatment of biological conditions including, but not limited to, vascular
complications of type I and type II diabetic-induced vasculopathies, other
vasculopathies, microangiopathies, renal insufficiency, Alzheimer's syndrome,
and inflammation-induced diseases such as atherosclerosis. Other inflammatory
related diseases include, but are not limited to, inflammatory diseases of the
joint
such as rheumatoid arthritis, osteoarthritis, autoimmune diseases such as
those
taught supra, streptococcal cell-wall induced arthritis, adjuvant-induced
arthritis,
bursitis; inflammatory diseases of the thyroid such as acute, subacute and
chronic
thyroiditis, pelvic inflammatory disease, hepatitis; Inflammatory bowel
diseases
such as Crohn's disease and colitis; neuroinflammatory diseases such as
multiple
sclerosis, abscess, meningitis, encephalitis, and vasculitis; inflammatory
diseases
of the heart such as myocarditis, chronic obstructive pulmonary disease,
atherosclerosis, pericarditis; inflammatory diseases of the skin such as acute
inflammatory dermatoses (urticaria (hives), spongiotic dermatitis, erythema
multiforme (em minor), Stevens-Johnson syndrome (sjs, em major), toxic
epidermal necrolysis (ten) and chronic inflammatory dermatoses (psoriasis,
lichen planus, discoid lupus erythematosus, acne vulgaris); inflammatory
diseases
of the eye such as uveitis, allergic conjunctivitis, corneal inflammation,
intraocular inflammation, iritis; laryngitis and asthma.
The compounds of the present invention have utility in inhibiting
inflammation and/or its associated cell activation by glycated proteins or
AGE.
Pharmacological inhibition of AGE-induced cell activation provides the basis
for
therapeutic intervention in many diseases, notably in diabetic complications
and
Alzheimer's disease. Therapeutic approaches for inhibition of AGE-induced
inflammation include, but are not limited to, blocking the glycation of
proteins,
blocking AGE interactions with receptors and blocking AGE-induced signaling
or signaling-associated inflammatory responses.
At least one activity of some of the compounds of the present invention is
to block AGE effects by inhibiting AGE-induced signaling. The sequence of
these signaling events leading to inflammation are not clear, but inhibition
of
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these signaling events leads to reduced or no inflammatory results. Compounds
that block AGE-induced up-regulation of inflammatory molecules were
determined using screening assays. Other aspects of the present invention
comprise methods and compositions comprising compounds that block glycated
protein-induced inflammation. Some compounds may effect AGE formation or
AGE cross-linking.
At least one activity of some of the compounds of the present invention is
to block AGE effects by inhibiting reactions with receptors of AGE and such
activities are also contemplated by the methods of the present invention for
treatment of related pathologies. For example, RAGE, a known receptor for
AGE, is a therapeutic target. Blocking RAGE inhibited AGE-induced
inflammation. Prior to use of the compounds of the present invention, the
multiple functions of RAGE and possible long term side effects of accumulated
AGE in plasma, have prevented this method of treatment from being
implemented. However, using the methods and compositions of the present
invention, more specific inhibitory compounds can be used for treatments and
overcome the current problems with treatments that target receptors.
Compounds of the present invention that have at least the activity of
modulating inflammation activity are shown in TABLE 5. The compounds
shown in this Table have the activity of modulating inflammation activity as
measured by the assays taught herein. The inclusion of compounds in the
categories of the Tables disclosed herein are not to be seen as limiting, in
that
compounds included in such Tables have at least the activity shown for
inclusion
in the Table and may have more or other activities. Nor are the Tables to be
seen
as limiting in that these are the only compounds disclosed herein that have
that
activity, representative compounds are shown in the Tables that have at least
that
particular activity for inclusion in the Table. One or more compounds
disclosed
herein have at least an activity that has utility in treatment of disease
states.
Examples of compounds that show at least this activity and utility are
shown in the following formula:
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or an ene, a diene, a triene, or an yne derivative thereof; a saturated
derivative
thereof; a stereoisomer thereof; or a salt thereof;
wherein:
R' is in each occurrence independently selected from -H; linear or
branched alkyl with up to 10 carbon atoms; cycloalkyl with up to 10 carbon
atoms; aryl; or (CHZ)aCN, wherein x is an integer from 0 to 6;
E is CH or N;
n is an integer from 0 to 3;
X' is selected from -H, rn-F, m-Cl, rn-Br, m-I, m-CN, m-NOa, m-SOaR~,
m-SOZOR~, m-NC(O)R~, or o-F, or X' and X2 together is a fused benzene,
pyridine, or dioxane ring;
Xa is selected from -H, o-Cl, o-Br, o-CF3, o-Rl, p-ORS, p-SRI, p-NR~a, p
F, p-C1, p-Br, p-CF3, p-CN, p-C(O)ORS, p-NC(O)R~, p-(4-morpholinyl), orp-(4
methyl-1-piperizinyl);
AY' is a halogen, or A is NR' or O and Y' is selected from cycloalkyl
with up to 10 carbon atoms, cycloalkyl with up to 10 carbon atoms substituted
with R', linear or branched alkyl with up to 10 carbon atoms, CH2R~,
~~''~CHR~
~NR~
(CHR~)yOR~, wherein y is an integer from 1 to 6, ; or AYl together are
N
HZC/ (CHZ),~ ~ wherein x is an integer from 3 to 5; and
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HRH
NJ NR~
DYa is a halogen, or D is NR~ and Ya is selected from ~t' , ,
cycloalkyl with up to 10 carbon atoms, cycloalkyl with up to 10 carbon atoms
substituted with R', linear or branched alkyl with up to 10 carbon atoms,
CH2R~,
N
I
N
~ ~ H ~~ CH CF
H2C-(C 2)X, wherein x ~s an integer from 3 to 5, , 2 3,
(CHR~)ZZ~, wherein z is an integer from 1 to 6, and Z' is selected from NR~~,
_ O
O ~N~ ~N
H2C-(CH~),~~ wherein x is an integer from 3 to 5, ~ , or
~N~N ~ N ~z
or NYZRj together is selected from ~---~ , wherein Z' is
O
N
O~O
selected from R , C(O)R , C(O)OR , pyndmyl, aryl, , ,
~~CH~)qOR~
N
or ~ ~ , wherein q is an integer from 0 to 6.
Further examples of compounds that show at least this activity and utility
are presented in Table 5, where compound activity is also shown. The activity
scale used in Table 5 is as follows (numbers are inclusive): "++-E-t-"
represents
between 0 and about 25% of IL6 production compared to cells that did not
receive compound (or per cent of control IL6 production); "+++" represents
I S between about 25 and about 50% of control IL6 production; "++" represents
between about 50 and about 75% of control IL6 production; and "+" represents
between about 75 and 100% of control IL6 production. The note "n.d." indicates
that the activity of the compound was not determined in the given assay.
Further
note that any hydrogen atoms that are required for any atom to attain its
usual
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valence in a structure presented in Table 5, whether a carbon atom or a
heteroatom, should be inferred if it is not specifically indicated.
In addition to the above compounds, the compounds shown in Table 7,
and compositions comprising these compounds, also exhibit the activity of
modulating inflammation activity as measured by the assays taught herein. The
activity scale used in Table 7 is as follows (numbers are inclusive): "+++"
represents between about 85 to 100% inhibition of IL6 production in the
presence
of AGE or TNF, as compared to cells that did not receive any compound; "++"
represents between about 65 and about 85% inhibition of IL6 production in the
presence of AGE or TNF; and "+" represents between about 50 and about 65%
inhibition of IL6 production in the presence of AGE or TNF. As before, the
inclusion of compounds in the categories of the Tables disclosed herein are
not to
be seen as limiting, in that compounds included in such Tables have at least
the
activity shown for inclusion in the Tables and may have more or other
activities.
Nor are the Tables to be seen as limiting in that these are the only compounds
disclosed herein that have that activity, representative compounds are shown
in
the Tables that have at least that particular activity for inclusion in the
Table.
One or more compounds disclosed herein have at least an activity that has
utility
in treatment of disease states.
Enhanced formation and accumulation of glycated proteins and AGE are
thought to play a major role in the pathogenesis of diabetic complications,
and
atherosclerosis, leading to the development of a range of diabetic
complications
including nephropathy, retinopathy and neuropathy. There is ample in vivo
evidence that suggests that diabetes-related complications can be reduced by
1)
preventing glycation of proteins, 2) by breaking the cross-links in glycated
proteins or 3) by blocking glycated protein interaction with receptors.
Despite
the importance of AGE in the pathogenesis of diabetic microangiopathies, there
are no currently available medications known to block AGE formation.
Endothelium is the target organ of damage in diabetes. See Laight et al.,
15 DIABETES METAB. Res. REV. 274-82 (1999); Stehouwer et al., 34
CARDIOVASC. 55-68 (1997). Up-regulation of molecules involved in endothelial
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inflammation, such as IL-6 and monocyte chemoattractant protein-1 (MCP-1)
leads to endothelial dysfunction and vasculopathy. See Stehouwer et al., 34
CARDIOVASC. 55-68 (1997); Libby, 247 J. INTERN. MED. 349-58 (2000); Van
Lente, 293 CLINICA. CHIMICA. ACTH. 31-52 (2000).
IL-6 is a pro-inflammatory cytokine that is known to play a key role in the
pathogenesis of diabetes and atherosclerosis. See Horii et al., 39 KIDNEY INT.
SUPPL. 71-5 (1993); Huber et al., 19 ARTERIOSCLER THROMB. VASC. BIOL. 2364-
67 (1999); Shikano et al., 85 NEPHRON 81-5 (2000); Pickup et al., 8(67) LIFE
SCI.
291-300 (2000). IL-6 also promotes the growth of renal mesangial cells thus
contributing to nephropathy. See Kado et al., 36 ACTH. DIABETOL. 67-72 (1999).
The serum IL-6 level in diabetic subjects was significantly higher than in
normal
healthy controls (3.48 +/- 3.29 pg/ml vs 0.784 +/- 0.90 pg/ml, mean +/- SD).
In
addition the urinary IL-6 level is a good indicator of diabetic nephropathy.
Serum IL-6 is useful in the evaluation of atherosclerosis and nephropathy.
MCP-l, another pro-inflammatory cytokine is found highly expressed in
human atherosclerotic lesions and postulated to play a central in monocyte
recruitment into the arterial wall and developing lesions. See Libby, 247 J.
INTERN. MED. 349-58 (2000). Recent results show that MCP-1 is also a key
pathogenic molecule in diabetic nephropathy. See Eitner et al., 51 KIDNEY INT.
69-78 (1997); Banba et al. 58 KIDNEY INT. 684-90 (2000). Glycated albumin
stimulates endothelial production of IL-6 and MCP-1. The effects of glycated
albumin on IL-6 production are comparable to that of TNFa, a known inducer of
IL-6. These cytokines are known to be factors in vascular diseases.
The activity of the compounds of the present invention in inhibiting
glycated protein- and AGE-induced inflammation can be determined using the
assays described herein and in U.S. Patent Application No. 10/026,335, which
is
herein incorporated in its entirety. Such assays comprise measurement of the
specific activity of biological components involved in a known cellular
response.
The assays provide a measurable response in which the activity of the
compounds
is determined. One assay comprises measurement of the effects of compounds on
an inflammatory response by cells to the presence of a stimulating agent. Yet
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another assay comprises endothelial cells that are stimulated by the addition
of a
glycated protein, the stimulating agent. The endothelial cells respond by
producing specific cytokines. The amount of cytokines produced are determined
by measurement protocols known to those skilled in the art. The compounds of
the present invention are then added to the assay and the production of
cytokines
is measured. From the comparison of the assay without the compound with the
assay with the compound, the biological effect of the compound can be
determined. The compound may have an inhibitory effect, a stimulatory effect,
or no effect at all.
The amount and type of cytokine produced can be determined using
immunological methods, such as ELISA assays. The methods of the present
invention are not limited by the type of assay used to measure the amount of
cytokine produced, and any methods known to those skilled in the art and later
developed can be used to measure the amount of cytokines produced in response
to the stimulating agent and to the compound having unknown activity.
An aspect of the present invention comprises methods and compositions
for the treatment of diseases, preconditions or pathologies associated with
inflammatory cytokines and other inflammation related molecules including, but
not limited to IL-6, VCAM-l, AGE-induced MCP-l, (monocyte chemoattractant
protein 1), heme oxygenase, insulin-like growth factor, selectins, IP-10, MIG
and
I-TAC, NF-KB, IL-1 (3 (interleukin 1 (3), IL-11 (interleukin 11), m-CSF
(macrophage colony stimulating factor), fibrinogen, TNF-a (tumor necrosis
factor a), adhesion molecules, selectins, VCAM-1 (Vascular Cell Adhesion
Molecule-1), CRP (C-reactive .protein), and PAI-1 (plasminogen activator
inhibitor-1). .Examples of such diseases include the pathogenesis of
atherosclerosis and the development of diabetic vasculopathy in type II
diabetes.
For example, affecting the activity or level of TNFa is a key mediator of
tissue
damage following acute or chronic inflammatory reactions. The present
invention contemplates providing compositions and methods that modulate the
effects of cytokines and inflammatory molecules such as TNFa, IL-6, VCAM-1,
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IP-10, MIG, I-TAC and AGE-induced MCP-1, and treat the associated diseases,
acute or chronic conditions, preconditions and pathologies.
Assays for determining the activity of compounds capable of modulating
inflammation include those taught in U.S. Patent Application No. 10/026,335
and
09/969,013, which are both expressly incorporated by reference. In general,
once
the baseline response to the stimulating agent for the production of cytokines
by
the endothelial cells is established, thus comprising the control levels for
the
screening assay, the methods comprise addition of compounds of the present
invention. The effect of the compound on the baseline response is determined
by
comparing the amount of cytokine produced in the presence of the stimulating
agent and the amount of cytokine produced in the presence of the stimulating
agent and the compound of the present invention. In a preferred method,
compounds that have inhibitory effects on the inflammation of the cells in the
presence of glycated albumin are then used as therapeutic agents. One or more
compounds may be added to the screening assay. Combinations or mixtures of
compounds can be added. Different amounts and formulations of the compounds
are added to determine the effects on the screening assay. The screening assay
may also be used to determine stimulatory compounds or compounds that have
no effects in the assay.
The present invention comprises methods and compositions for the
treatment and prevention of disease, conditions and pathologies associated
with
inflammation. Such methods comprise administration of compositions
comprising compounds capable of modulating the activity of molecules
associated with inflammation such as AGE or cytokines or other cellular
factors,
including release rates or activity, and include compositions comprising
compounds disclosed herein with inflammation modulating activity.
Administration of such compounds that are effective in modulating inflammation
are administered to humans and animals suspected of having or who have
inflammatory diseases, for example, diabetic-induced vasculopathies,
autoimmune diseases, renal insufficiency, Alzheimer's syndrome, and
inflammation-induced diseases such as atherosclerosis. Effective amounts are
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administered to such humans and animals in dosages that are safe and
effective,
including, but not limited to, the ranges taught herein. Routes of
administration
include, but are not limited to, those disclosed herein. As disclosed herein,
compositions comprising such compounds may be used in conjunction with other
therapeutic agents or in methods comprising steps such as altered patient
activities, including, but not limited to, changes in exercise or diet.
VI. Cytotoxic Activity
An embodiment of the present invention comprises methods and
compositions comprising compounds that have at least the activity of causing
cellular death or a cessation of cellular activity, referred to herein as
cytotoxic
activity. This activity can be used in methods for i~ vitro or in vivo
cytotoxicity.
For example, compounds having this activity can be selectively delivered to an
area within a living organism to selectively kill cells in that area. Such
methods
I S are using in treating hyperproliferative cells, such as cancers, or other
unwanted
cellular growth or cellular activities. One aspect of the invention provides
compositions comprising compounds that nonselectively kill cells. Another
aspect of the invention provides compounds that selectively kill cells, for
example, cells that have a particular cellular marker or other identifying
characteristic such as metabolic rate or uptake of a particular compound, such
as
sodium, calcium or thymidine.
The present invention also provides compositions for and methods of
treatment of biological conditions including, but not limited to, conditions
for
which cytotoxic activity is a treatment. For example, the compositions and
methods for providing compounds that have at least the activity of
cytotoxicity
are useful in the treatment or prophylaxis of at least one hyperproliferative
disease in a cell, tissue, organ, animal, or patient including, but not
limited to,
malignant and non-malignant cell growth, leukemia, acute leukemia, acute
lymphoblastic leukemia (ALL), B-cell, T-cell or FAB ALL, acute myeloid
leukemia (AML), chromic myelocytic leukemia (CML), chronic lymphocytic
leukemia (CLL), hairy cell leukemia, myelodyplastic syndrome (MDS), a
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lymphoma, Hodgkin's disease, a malignamt lymphoma, non-hodgkin's
lymphoma, Burlcitt's lymphoma, multiple myeloma, I~aposi's sarcoma, colorectal
carcinoma, pancreatic carcinoma, nasopharyngeal carcinoma, malignant
histiocytosis, paraneoplastic syndrome/hypercalcemia of malignancy, solid
tumors, adenocarcinomas, sarcomas, malignant melanoma, hemangioma,
metastatic disease, cancer related bone resorption, cancer related bone pain,
and
the like.
Compounds of the present invention that have at least the activity of
cytotoxicity are shown in TABLE 4A and B. The compounds shown in this
Table have the activity of cytotoxicty as measured by the assays taught
herein.
The inclusion of compounds in the categories of the Tables disclosed herein
are
not to be seen as limiting, in that compounds included in such Tables have at
least the activity shown for inclusion in the Table and may have more or other
activities. Nor are the Tables to be seen as limiting in that these are the
only
compounds disclosed herein that have that activity, representative compounds
are
shown in the Tables that have at least that particular activity for inclusion
in the
Table. One or more compounds disclosed herein have at least an activity that
has
utility in treatment of disease states.
Examples of compounds that show at least this activity and utility are
shown in the following formula:
A
or an ene, a diene, a triene, or an yne derivative thereof; a saturated
derivative
thereof; a stereoisomer thereof; or a salt thereof;
wherein:
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R' is in each occurrence independently selected from -H; linear or
branched alkyl with up to 10 carbon atoms; cycloalkyl with up to 10 carbon
atoms; or aryl;
E is CH or N;
n is an integer from 0 to 3;
X' is selected from -H, rn-F, m-Cl, m-Br, na-I, nr-CN, m-NOZ, rrt-S02R',
or rrr-SOZOR', or X' and X2 together is a fused benzene or pyridine ring;
XZ is selected from -H, o-CI, o-Br, p-OR', p-SR', p-NR'a, p-F, p-Cl, p-Br,
p-CF3, p-C(O)OR', p-OM, or p-SM, wherein M is selected from Li, Na, K, Mg,
or Ca;
A is selected from NR' or O, wherein Y' is selected from cycloalkyl with
up to 10 carbon atoms, linear or branched alkyl with up to 10 carbon atoms, or
.""",n",
/ ~ X~
I
\\X2 when A is NR', and wherein Y' is selected from R' or CHaR' when A
N N
~~ or ~~ ; and
is O; or AY is selected from a halogen, ,
NJ
I S DYa is a halogen, or D is NR' and Y2 is selected from ~' ,
~~CHR~
~NR~
or (CHR')XNR'2, wherein x is an integer from 1 to 6.
Additional examples of compounds that show at least this activity and
utility are shown in the following formula:
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X1
R~ \
\ 2
Hue., \ nip
R1
1
or an ene, a dime, a triene, or an yne derivative thereof; a saturated
derivative
thereof; a stereoisomer thereof; or a salt thereof;
wherein:
R' is in each occurrence independently selected from H; linear or
branched alkyl with up to 10 carbon atoms; or cycloalkyl with up to 10 carbon
atoms;
X' is in each occurrence independently selected from -H, m-F, m-CI, m-
Br, m-I, m-CN, m-NO2, m-S02R~, or m-SOZOR~;
Xa is in each occurrence independently selected from o-CH3, p-ORI, p-
SR~, p NR~2, or p-OM or p-SM, wherein M is selected from Li, Na, I~, Mg, or
Ca;
Y' is selected from cycloalkyl with up to 10 carbon atoms;
(CH2)n
~~J
N
wherein n is 1 or 2; or X2 ; and
~~CHR~
NR~
N
Y2 is selected from R1 or
Further examples of compounds that show at least this activity and utility
are presented in Tables 4A and 4B. The compound nomenclature of Tables 4A
and 4B, as in the other tables presented herein, was generated using Autonom,
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where the name provided may be the Beilstein or CAS version of the chemical
name. Note that any hydrogen atoms that are required for any atom to attain
its
usual valence in a structure presented in Tables 4A and 4B, whether a carbon
atom or a heteroatom, should be inferred if it is not specifically indicated.
Assays for determining the activity of compounds capable of cytotoxic
activity include those taught in herein and others that are well known in the
art.
In general, to determine if there is cytotoxic activity associated with a
compound,
cells of a particular type, in a growing stage or a quiescient stage, are
treated with
the compound of interest. Various parameters of cell death or cessation are
used
to measure the effects of the compound. For example, the amount of nucleic
acid
or protein synthesis can be measured or visual observation of the state of the
cells, such as release from the substrate, can be used to measure the state of
the
cel Is.
The present invention comprises methods and compositions for the
treatment and prevention of diseases or conditions that present or result from
cellular proliferation or unwanted cellular growth or cellular activity. Such
methods comprise administration of compositions comprising compounds
capable of modulating cellular activity or causing cellular death or cessation
of
growth such as compositions comprising compounds disclosed herein that have
cytotoxic activity. Administration of such compounds that are effective in
cytotoxic activity are administered to humans and animals suspected of having
or
who have, for example, cancer, overactive tissues such as thyroid or
hypothalamus, or cellular conditions where factors are released in unwanted
amounts. Effective amounts are administered to such humans and animals in
dosages that are safe and effective, including, but not limited to, the ranges
taught
herein. Routes of administration include, but are not limited to, those
disclosed
herein. As disclosed herein, compositions comprising such compounds may be
used in conjunction with other therapeutic agents or in methods comprising
steps
such as altered patient activities.
Compound/Composition-Coated Medical Devices
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The compounds of the present invention may be used alone or in
combination with other agents along with delivery devices to effectively
prevent
and treat the diseases described herein, though particular applications are
found
in vascular disease, and in particular, vascular disease caused by injury
and/or by
transplantation. Though this example focuses on vascular disease, provision of
the compounds of the present invention with medical devices for treatment of
the
diseases and conditions capable of being treated with the compounds is
contemplated by the present invention.
Various medical treatment devices utilized in the treatment of vascular
disease may ultimately induce further complications. For example, balloon
angioplasty is a procedure utilized to increase blood flow through an artery
and is
the predominant treatment for coronary vessel stenosis. However, the procedure
typically causes a certain degree of damage to the vessel wall, thereby
creating
new problems or exacerbating the original problem at a point later in time.
Although other procedures and diseases may cause similar injury, exemplary
embodiments of the present invention will be described with respect to the
treatment of restenosis and related complications following percutaneous
transluminal coronary angioplasty and other similar arterial/venous
procedures,
including the joining of arteries, veins and other fluid carrying conduits in
other
organs or sites of the body, such as the liver, lung, bladder, kidney, brain,
prostate, neck and legs.
The local delivery of a compound of the present invention and, in some
embodiments, along with other therapeutic agents, from a stmt prevents vessel
recoil and remodeling through the scaffolding action of the stmt. The activity
of
compound provided, with or without other therapeutic agents, helps determine
for
which application, to treat which disease, the coated medical device is being
administered. For example, compound-coated stems can prevent multiple
components of neointimal hyperplasia or restenosis as well as reduce
inflammation and thrombosis. Local administration of a compound of the present
invention and other therapeutic agents to stented coronary arteries may also
have
additional therapeutic benefit. For example, higher tissue concentrations of
the
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compounds of the present invention and other therapeutic agents may be
achieved
utilizing local delivery rather than systemic administration. In addition,
reduced
systemic toxicity may be achieved utilizing local delivery rather than
systemic
administration while maintaining higher tissue concentrations. In utilizing
local
delivery from a scent rather than systemic administration, a single procedure
may
suffice with better patient compliance. An additional benefit of combination
therapeutic agent and/or compound therapy may be to reduce the dose of each of
the therapeutic agents, thereby limiting toxicity, while still achieving a
reduction
in restenosis, inflammation and thrombosis. Local stmt-based therapy is
therefore a means of improving the therapeutic ratio (efficacy/toxicity) of
anti-
restenosis, anti-inflammatory, and anti-thrombotic therapeutic agents.
Although exemplary embodiments of the invention will be described with
respect to the treatment of restenosis and other related complications, it is
important to note that the local delivery of a compound of the present
invention,
I S alone or as part of a therapeutic agent combination, may be utilized to
treat a
wide variety of conditions utilizing any number of medical devices, or to
enhance
the function and/or life of the device. For example, intraocular lenses,
placed to
restore vision after cataract surgery is often compromised by the formation of
a
secondary cataract. The latter is often a result of cellular overgrowth on the
lens
surface and can be potentially minimized by combining one or more compounds
of the present invention having activity that is effecting in proventing
unwanted
cellular growth with the device. Other medical devices that often fail due to
tissue in-growth or accumulation of proteinaceous material in, on and around
the
device, such as shunts for hydrocephalus, . dialysis grafts, colostomy bag
attachment devices, ear drainage tubes, leads for pace makers and implantable
defibrillators can also benefit from the combinations of the compounds of the
present invention, possibly other pharmaceutical agents, and the devices..
Other
surgical devices, sutures, staples, anastornosis devices, vertebral disks,
bone pins,
suture anchors, hemostatic barriers, clamps, screws, plates, clips, vascular
implants, tissue adhesives and sealants, tissue scaffolds, various types of
dressings, bone substitutes, intraluminal devices, and vascular supports could
also
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provide enhanced patient benefit using this compound-device combination
approach. Essentially, any type of medical device may be coated in some
fashion
with at least one compound of the present invention, alone or as part of a
therapeutic agent combination, which enhances treatment over the use of the
device or therapeutic agent without combination with the compound..
As disclosed supra, the compounds of the present invention can be
administered in combinational therapies with other therapeutic agents, and are
not
limited to only the other therapeutic agents disclosed herein. Thus, the
present
invention also contemplates, in addition to various medical devices, the
coatings
on these devices may be used to deliver a compound of the present invention in
combination with other therapeutic agents. This illustrative list of
therapeutic
agents can be administered through pharmeutical means or in association with
medical devices and such therapeutic agents include, but are not limited to,
antiproliferative/antimitotic agents including natural products such as vinca
IS alkaloids (e.g., vinblastine, vincristine, and vinorelbine), paclitaxel,
epidipodophyllotoxins (e.g., etoposide, teniposide), antibiotics (dactinomycin
(actinomycin D) daunorubicin, doxorubicin and idarubicin), anthracyclines,
mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin, enzymes
(L-asparaginase which systemically metabolizes L-asparagine and deprives cells
which do not have the capacity to synthesize their own asparagine);
antiplatelet
agents such as G(GP) IIb/IIIa inhibitors and vitronectin receptor antagonists;
antiproliferative/antimitotic alkylating agents such as nitrogen mustards
(mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil),
ethylenimines and methylmelamines (hexamethyhnelamine and thiotepa), alkyl
sulfonates-busulfan, nirtosoureas (carmustine (BCNU) and analogs,
streptozocin), trazenes-dacarbazinine (DTIC); antiproliferative/antimitotic
antimetabolites such as folic acid analogs (methotrexate), pyrimidine analogs
(fluorouracil, floxuridine, and cytarabine), purine analogs and related
inhibitors
(mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxyadenosine
(cladribine)); platinum coordination complexes (cisplatin, carboplatin),
procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones (e.g.
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estrogen); anticoagulants (heparin, synthetic heparin salts and other
inhibitors of
thrombin); fibrinolytic agents (such as tissue plasminogen activator,
streptokinase
and urolcinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab;
antimigratory; antisecretory (breveldin); anti-inflammatory agents such as
adrenocortical steroids (cortisol, cortisone, fludrocortisone, prednisone,
prednisolone, 6a-methylprednisolone, triamcinolone, betamethasone, and
dexamethasone), non-steroidal agents (salicylic acid derivatives, i.e.,
aspirin;
para-aminophenol derivatives, i.e., acetominophen; indole and indene acetic
acids
(indomethacin, sulindac, and etodalac), heteroaryl acetic acids (tolmetin,
diclofenac, and ketorolac), arylpropionic acids (ibuprofen and derivatives),
anthranilic acids (mefenamic acid, and meclofenamic acid), enolic acids
(piroxicam, tenoxicam, phenylbutazone, and oxyphenthatrazone), nabumetone,
gold compounds (auranofin, aurothioglucose, gold sodium thiomalate);
immunosuppressives. (Cyclosporine, tacrolimus (FIB-506), sirolimus
(rapamycin), azathioprine, mycophenolate mofetil); angiogenic agents: vascular
endothelial growth factor (VEGF), fibroblast growth factor (FGF); angiotensin
receptor blockers; nitric oxide donors; anti-sense oligionucleotides and
combinations thereof; cell cycle inhibitors, mTOR inhibitors, and growth
factor
signal transduction kinase inhibitors.
Although any number of stents may be utilized in accordance with the
present invention, for simplicity, a limited number of stems will be described
in
exemplary embodiments of the present invention. The skilled artisan will
recognize that any number of stems may be utilized in connection with the
present invention. In addition, as stated above, other medical devices may be
utilized. For example, though stems are described, sleeves outside the vessels
are
also contemplated, as are other medical devices that can provide a substrate
for
administration for at least one of the compounds of the present invention.
A stmt is commonly used as a tubular structure left inside the lumen of a
duct to relieve an obstruction. Typically, stems are inserted into the lumen
in a
non-expanded form and are then expanded autonomously, or with the aid of a
second device in situ. A common method of expansion occurs~through the use of
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a catheter-mounted, angioplasty balloon that is inflated within the stenosed
vessel
or body passageway in order to shear and disrupt the obstructions associated
with
the wall components of the vessel and to obtain an enlarged lumen.
A stmt may resemble an expandable cylinder and may comprise a
fenestrated structure for placement in a blood vessel, duct or lumen to hold
the
vessel, duct or lumen open, more particularly for protecting a segment of
artery
from restenosis after angioplasty. The stmt may be expanded circumferentially
and maintained in an expanded configuration that is circumferentially or
radially
rigid. The stent may be axially flexible and when flexed at a band, for
example,
the stmt avoids any externally protruding component parts.
The stmt may be fabricated utilizing any number of methods. For
example, the stmt may be fabricated from a hollow or formed stainless steel
tube
that may be machined using lasers, electric discharge milling, chemical
etching or
other means. The stent is inserted into the body and placed at the desired
site in
an unexpanded form. In one embodiment, expansion may be effected in a blood
vessel by a balloon catheter, where the final diameter of the stmt is a
function of
the diameter of the balloon catheter used. It should be appreciated that a
stmt in
accordance with the present invention may be embodied in a shape-memory
material including, for example, an appropriate alloy of nickel and titanium
or
stainless steel.
Structures formed from stainless steel may be made self expanding by
configuring the stainless steel in a predetermined manner, for example, by
twisting it into a braided configuration. In this embodiment, after the stmt
has
been formed it may be compressed so as to occupy a space sufficiently small as
to permit its insertion in a blood vessel or other tissue by insertion means,
wherein the insertion means include a suitable catheter, or flexible rod. Upon
emerging from the catheter, the stent may be configured to expand into the
desired configuration where the expansion is automatic or triggered by a
change
in pressure, temperature or electrical stimulation.
Furthermore, a stmt may be modified to comprise one or more reservoirs.
Each of the reservoirs may be opened or closed as desired. These reservoirs
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be specifically designed to hold the the compound or compound/therapeutic
agent
combination to be delivered. Regardless of the design of the stmt, it is
preferable
to have the compound or compound/therapeutic agent combination dosage
applied with enough specificity and a sufficient concentration to provide an
effective dosage in the affected area. In this regard, the reservoir size in
the
bands is preferably sized to adequately apply the the compound or
compound/therapeutic agent combination dosage at the desired location and in
the desired amount.
In an alternative embodiment, the entire inner and outer surface of the
stmt may be coated with the compound or compound/therapeutic agent
combination in therapeutic dosage amounts. The coating techniques may vary
depending on the the compound or compound/therapeutic agent combination.
Also, the coating techniques may vary depending on the material comprising the
stmt or other intraluminal medical device.
One or more compounds of the present invention and, in some instances,
other therapeutic agents as a combination, may be incorporated onto or affixed
to
the stent in a number of ways. In one embodiment, the compound is directly
incorporated into a polymeric matrix and sprayed onto the outer surface of the
stmt. The compound elutes from the polymeric matrix over time and enters the
surrounding tissue. The compound preferably remains on the stmt for at least
three days up to approximately six months, and more preferably between seven
and thirty days.
Any number of non-erodible polymers may be utilized in conjunction
with the compound, and such polyermic compositions are well known in the art.
In one embodiment, the polymeric matrix comprises two layers. The base layer
comprises a solution of polyethylene-covinylacetate) and
polybutylmethacrylate.
The compound is incorporated into this base layer. The outer layer comprises
only polybutylmethacrylate and acts as a diffusion barrier to prevent the
compound from eluting too quickly. The thickness of the outer layer or topcoat
determines the rate at which the compound elutes from the matrix. Essentially,
the compound elutes from the matrix by diffusion through the polymer matrix.
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Polymers are permeable, thereby allowing solids, liquids and gases to escape
therefrom. The total thickness of the polymeric matrix is in the range from
about
one micron to about twenty microns or greater. It is important to note that
primer
layers and metal surface treatments may be utilized before the polymeric
matrix
is affixed to the medical device. For example, acid cleaning, alkaline (base)
cleaning, salinization and parylene deposition may be used as part of the
overall
process described above.
The polyethylene-co-vinylacetate), polybutylmethacrylate and compound
solution may be incorporated into or onto the stmt in a number of ways. For
example, the solution may be sprayed onto the stmt or the stmt may be dipped
into the solution. Other methods include spin coating and plasma
polymerization.
In one embodiment, the solution is sprayed onto the stmt and then allowed to
dry. In another embodiment, the solution may be electrically charged to one
polarity and the stmt electrically charged to the opposite polarity. In this
manner,
the solution and stmt will be attracted to one another. In using this type of
spraying process, waste may be reduced and more precise control over the
thickness ofthe coat may be achieved.
Drug-coated stents are manufactured by a number of companies including
Johnson & Johnson, Inc. (New Brunswick, NJ), Guidant Corp. (Santa Clara, CA),
Medtronic, Inc. (Minneapolis, MN), Cook Group Incorporated (Bloomington,
IN), Abbott Labs., Inc. (Abbott Park, IL), and Boston Scientific Corp.
(Natick,
MA). See e.g., U.S. Patent No. 6,273, 913; U.S. Patent Application No.
20020051730; WO 02126271; and WO 02/26139, each expressly entirely
incorporated herein by reference.
Expression Profiles and Microarray Methods of Use
Other aspects of the present invention comprise compositions and
methods for microarray devices. Such microarray devices and methods comprise
a variety of microarrays that may be used, for example, to study and monitor
gene expression in response to treatment with the compounds of the present
invention. The microarrays may comprise nucleic acid sequences, carbohydrates
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or proteins that are determinative for specific cells, tissues, species,
disease states,
prognoses, disease progression, or any other combination of molecules that can
be used to determine an effect of one ~or more of the compounds of the present
invention
For example, the microarrays of the present invention may be derived
from, or representative of, for example, a specific organism or cell type,
including human microarrays, vascular microarrays, inflammation microarrays,
cancer microarrays, apoptosis microarrays, oncogene and tumor suppressor
microarrays, cell-cell interaction microarrays, cytokine and cytokine receptor
microarrays, blood microarrays, cell cycle microarrays, neuroarrays, mouse
microarrays, and rat microarrays, or combinations thereof. In further
embodiments, the microarrays may represent diseases including cardiovascular
diseases, vasculopathic conditions, inflammatory diseases, autoimmune
diseases,
neurological diseases, immunoIogical diseases, various cancers, infectious
diseases, endocrine disorders, and genetic diseases.
Alternatively, the microarrays useful in assessing the efficacy of the
compounds of the present invention may represent a particular tissue type
including, but not limited to, heart, liver, prostate, lung, nerve, muscle, or
connective tissue; preferably coronary artery endothelium, umbilical artery
endothelium, umbilical vein endothelium, aortic endothelium, dermal
microvascular endothelium, pulmonary artery endothelium, myometrium
microvascular endothelium, keratinocyte epithelium, bronchial epithelium,
mammary epithelium, prostate epithelium, renal cortical epithelium, renal
proximal tubule epithelium, small airway epithelium, renal epithelium,
umbilical
artery smooth muscle, neonatal dermal fibroblast, pulmonary artery smooth
muscle, dermal fibroblast, neural progenitor cells, skeletal muscle,
astrocytes,
aortic smooth muscle, mesangial cells, coronary artery smooth muscle,
bronchial
smooth muscle, uterine smooth muscle, lung fibroblast, osteoblasts, prostate
stromal cells, or combinations thereof.
The present invention further contemplates microarrays comprising a
gene expression profile comprising one or more polynucleotide sequences
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including complementary and homologous sequences, wherein said gene
expression profile is generated from a cell type treated with a compound of
the
present invention and is selected from the group comprising coronary artery
endothelium, umbilical artery endothelium, umbilical vein endothelium, aortic
endothelium, dermal microvascular endothelium, pulmonary artery endothelium,
myometrium microvascular endothelium, keratinocyte epithelium, bronchial
epithelium, mammary epithelium, prostate epithelium, renal cortical
epithelium,
renal proximal tubule epithelium, small airway epithelium, renal epithelium,
umbilical artery smooth muscle, neonatal dermal fibroblast, pulmonary artery
smooth muscle, dermal fibroblast, neural progenitor cells, skeletal muscle,
astrocytes, aortic smooth muscle, mesangial cells, coronary artery smooth
muscle, bronchial smooth muscle, uterine smooth muscle, lung fibroblast,
osteoblasts, and prostate stromal cells.
The present invention contemplates microarrays comprising one or more
protein-binding agents, wherein a protein expression profile is generated from
a
cell type treated with a compound of the present invention and is selected
from
the group comprising coronary artery endothelium, umbilical artery
endothelium,
umbilical vein endothelium, aortic endothelium, dermal microvascular
endothelium, pulmonary artery endothelium, myometrium microvascular
endothelium, keratinocyte epithelium, bronchial epithelium, mammary
epithelium, prostate epithelium, renal cortical epithelium, renal proximal
tubule
epithelium, small airway epithelium, renal epithelium, umbilical artery smooth
muscle, neonatal dermal fibroblast, pulmonary artery smooth muscle, dermal
fibroblast, neural progenitor cells, skeletal muscle, astrocytes, aortic
smooth
muscle, mesangial cells, coronary artery smooth muscle, bronchial smooth
muscle, uterine smooth muscle, lung fibroblast, osteoblasts, and prostate
stromal
cells.
More specifically, the present invention contemplates methods for the
reproducible measurement and assessment of the expression of specific mRNAs
or proteins in, for example, a specific set of cells. One method combines and
utilizes the techniques of laser capture microdissection, T7-based RNA
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amplification, production of cDNA from amplified RNA, and DNA microarrays
containing immobilized DNA molecules for a wide variety of specific genes,
including HSPGs such as perlecan, to produce a profile of gene expression
analysis for very small numbers of specific cells. The desired cells are
individually identified and attached to a substrate by the laser capture
technique,
and the captured cells are then separated from the remaining cells. RNA is
then
extracted from the captured cells and amplified about one million-fold using
the
T7-based amplification technique, and cDNA may be prepared from the
amplified RNA. A wide variety of specific DNA molecules are prepared that
hybridize with specific polynucleotides of the microarray, and the DNA
molecules are immobilized on a suitable substrate. The cDNA made from the
captured cells is applied to the microarray under conditions that allow
hybridization of the cDNA to the immobilized DNA on the microarray. The
expression profile of the captured cells is obtained from the analysis of the
hybridization results using the amplified RNA or cDNA made from the amplified
RNA of the captured cells, and the specific immobilized DNA molecules on the
microarray. The hybridization results demonstrate, for example, which genes of
those represented on the microarray as probes are hybridized to cDNA from the
captured cells, and/or the amount of specific gene expression. The
hybridization
results represent the gene expression profile of the captured cells. The gene
expression profile of the captured cells can be used to compare the gene
expression profile of a different set of captured cells. For example, gene
expression profiles may be generated from cells treated (and not treated) with
a
compound of the present invention. The .similarities and differences provide
useful information for determining the differences between the same cell type
under different conditions, more specifically, the change in gene expression
in
response to treatment with a compound of the present invention.
The techniques used for gene expression analysis are likewise applicable
in the context of protein expression profiles. Total protein may be isolated
from a
cell sample and hybridized to a microarray comprising a plurality of protein
binding agents, which may include antibodies, receptor proteins, small
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and the like. Using any of several assays known in the art, hybridization may
be
detected and analyzed as described above. In the case of fluorescent
detection,
algorithms may be used to extract a protein expression profile representative
of
the particular cell type. In this regard, the change in protein expression in
response to treatment of cells with a compound of the present invention may be
evaluated.
Thus, in one aspect, the present invention comprises at least one
microarray corresponding to a population of genes isolated from a particular
tissue or cell type in methods that is used to detect changes in gene
transcription
levels that result from exposing the selected tissue or cells to at least one
compound of the present invention. In this embodiment, a biological sample
derived from an organism, or an established cell line, may be exposed to at
least
one compound of the present invention in vivo or ex vivo. Thereafter, the gene
transcripts, primarily mRNA, of the tissue or cells are isolated by methods
well-
known in the art. SAMBROOIC ET AL., MOLECULAR CLONING: A LAB. MANUAL
(2001). The isolated transcripts are then contacted with a microarray under
conditions where the transcripts hybridize with a corresponding probe to form
hybridization pairs. Thus, the microarray provides a model of the
transcriptional
responsiveness following exposure to at least one compound of the present
invention. Such information can be used to determine therapeutic candidates. A
hybridization signal may then be detected at each hybridization pair to obtain
a
gene expression profile.
Gene and/or protein expression profiles and microarrays may also be used
to identify activating or non-activating compounds of a particular gene such
as
perlecan or other HSPG. Compounds that increase transcription rates or
stimulate, maintain, or stabilize the activity of a protein are considered
activating,
and compounds that decrease rates or inhibit the activity of a protein are non-
activating. Moreover, the biological effects of a compound may be reflected in
the biological state of a cell. This state is characterized by the cellular
constituents. One aspect of the biological state of a cell is its
transcriptional state.
The transcriptional state of a cell includes the identities and amounts of the
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constituent RNA species, especially mRNAs, in the cell under a given set of
conditions. Thus, the gene expression profiles, microarrays, and algorithms
discussed herein may be used to analyze and characterize the transcriptional
state
of a given cell or tissue following exposure to an activating or non-
activating
compound, specifically, a compound of the present invention.
Microarray techniques and methods for analyzing results are well known
in the art. See U.S. Patent Nos. 6,263,287; 6,239,209; 6,218,122; 6,197,599;
6,156,501; 5,874,219; 5,837,832; 5,700,637; 5,445,934; U.S. Patent Application
Nos. 200110014461 A1; 2001/0039016 Al; 2001/0034023 A1; WO 01/94946;
and WO 01177668. See also, Haab et al., 2 GENOME BIOLOGY 1-12 (2001);
Brown et al., 97 PROC. NATL. ACAD. SCI. USA 262-7 (2000); Getz et al., 97
PROC. NATL. ACAD. SCI. USA 12079-84 (2000); Harrington et al., 3 CURRENT
OPINION MICROBIOL 285-91 (2000); Holter et al., 97 PROC. NATL. ACAD. SCI.
USA 8409-14 (2000); MacBeath et al., 289 SCIENCE 1760-63 (2000); Duggan et
al., 21 NATURE GENET 10-14 (1999); Lipshutz et al., 21 NATURE GENET 5-9
(1999); Eisen et al., 95 PROC. NATL. ACRD. SCI. USA 14863-68 (1998);
Ermolaeva et al., 20 NATURE GENET. 19-23 (1998); Hacia et al., 26 NUCLEIC
ACIDS RES. 3865-66 (1998); Lockhart et al., NUCLEIC ACIDS SYMP. SER. 11-12
(1998); Schena et al., 16 TRENDS BIOTECHNOL. 301-6 (1998); Shalom 46
PATROL. BIOL. 107-9 (I 998) ; Welford et al., 26 NUCLEIC ACID RES. 3059-65
(1998); Blanchard et al., I 1 BIOSENSORS BIOELECTRONICS 687-90 (1996);
Lockhart et al., 14 NATURE BIOTECHNOL. 1675-80 (1996); Schena et al., 93
PROC. NATL. ACRD. SCI. USA 10614-19 (1996); Tomayo et al., 96 PROC. NATL.
ACAD. SCI. USA 2907-12 (1996); Schena et al., 270 SCIENCE 467-70 (1995)
Database Creation, Database Access and Associated Methods of Use
Another embodiment of the present invention comprises a variety of
methods for managing or using data related to the compounds, methods of
making the compounds, methods of using and administering the compounds, and
diagnosing, prognosing and following the outcomes associated with diseases in
which the compounds are effective in treating. For example, methods for
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providing diagnostics and predictors relating to biomolecules including HSPGS,
particularly, perlecan, are contemplated by the present invention. Also within
the
scope of this invention are methods providing diagnostics and predictors
relating
to the efficacy of the compounds of the present invention. The present
invention
further contemplates methods of providing expression profile databases, and
methods for producing such databases, for normal and diseased tissues.
The expression profile database may be an internal database designed to
include annotation information about the expression profiles generated to
assess
the effect of the compounds of the present invention and through other sources
and methods. Such information may include, for example, the databases in which
a given biomolecule was found, patient information associated with the
expression profile, including age, cancer or tumor type or progression,
information related to a compound of the present invention such as dosage and
administration information, descriptive information about related cDNAs
associated with the sequence, tissue or cell source, sequence data obtained
from
external sources, expression profiles for a given gene and the related disease
state
or course of disease, for example whether the expression profile relates to or
signifies a particular disease state, and preparation methods. The expression
profiles may be based on protein and/or palynucleotide microarray data
obtained
from publicly available or proprietary sources. The database may be divided
into
two sections: one for storing the sequences and related expression profiles
and
the other for storing the associated information. This database may be
maintained as a private database with a firewall within the central computer
facility. However, this invention is not so limited and the expression profile
database may be made available to the public.
The database may be a network system connecting the network server
with clients. The network may be any one of a number of conventional network
systems, including a local area network (LAN) or a wide area network (WAN), as
is known in the art (e.g., Ethernet). The server may include software to
access
database information for processing user requests, and to provide an interface
for
serving information to client machines. The server may support the World Wide
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Web and maintain a website and Web browser for client use. Clientlserver
environments, database servers, and networks are well documented in the
technical, trade, and patent literature.
Through the Web browser, clients may construct search requests for
retrieving data from, for example, a microarray database and an expression
profile database. For example, the user may "point and click" to user
interface
elements such as buttons, pull down menus, and scroll bars. The client
requests
may be transmitted to a Web application that formats them to produce a query
that may be used to gather information from the system database, based, for
example, on microarray or expression data obtained by the client, and/or other
phenotypic or genotypic information. Specifically, the client may submit
expression data based on microarray expression profiles obtained from a
patient
treated with a compound of the present invention and use the system to obtain
a
diagnosis based on that information based on a comparison by the system of the
l 5 client expression data with the expression data contained in the database.
By way
of example, the system compares the expression profiles submitted by the
client
with expression profiles contained in the database and then provides the
client
with diagnostic information based on the best match of the client expression
profiles with the database profiles. Thus, in one aspect, the comparison of
expression profiles aids the clinician in determining the effectiveness of
treatment
with a compound of the present invention.. Based on such a comparison, the
clinician may alter ar adjust the treatment regimen.
In addition, the website may provide hypertext links to public databases
such as GenBank and associated databases maintained by the National Center for
Biotechnology Information (NCBI), part of the National Library of Medicine as
well as, any links providing relevant information for gene expression
analysis,
genetic disorders, scientific literature, and the like. Information including,
but
not limited to, identifiers, identifier types, biomolecular sequences, common
cluster identifiers (GenBank, Unigene, Incyte template identifiers, and so
forth)
and species names associated with each gene, is contemplated.
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The present invention also provides a system for accessing and comparing
bioinformation, specifically expression profiles and other information which
is
useful in the context of the compositions and methods of the present
invention.
In one embodiment, the computer system may comprise a computer processor,
suitable memory that is operatively coupled to the computer processor, and a
computer process stored in the memory that executes in the computer processor
and which comprises a means for matching an expression profile of a
biomolecular sequence from a patient with expression profile and sequence
identification information of biomolecular sequences in a database. More
specifically, the computer system is used to match an expression profile
generated from a biological sample treated with a compound of the present
invention with expression profile and other information in a database.
Furthermore, the system for accessing and comparing information
contained in biomolecular databases comprises a computer program comprising
computer code providing an algorithm for matching an expression profile
generated from a patient, for example, treated with a compound of the present
invention, with expression profile and sequence identification information of
biomolecular sequences in a biomolecular database.
The present invention contemplates, in one embodiment, the use of a
Graphical User Interface ("GUI") for the access of expression profile
information
stored in a biomolecular database. In a specific embodiment, the GUI may be
composed of two frames. A first frame may contain a selectable list of
biomolecular databases accessible by the user. When a biomolecular database is
selected in the first frame, a second frame may display information resulting
from
the pair-wise comparison of the expression profile database with the client-
supplied expression profile as described above, along with any other
phenotypic
or genotypic information.
The second frame of the GUI may contain a listing of biomolecular
sequence expression information and profiles contained in the selected
database.
Furthermore, the second frame may allow the user to select a subset, including
all
of the biomolecular sequences, and to perform an operation on the list of
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biomolecular sequences. In one embodiment, the user may select the subset of
biomolecular sequences by selecting a selection box associated with each
biomolecular sequence. In another embodiment, the operations that may be
performed include, but are not limited to, downloading all listed biomolecular
sequences to a database spreadsheet with classification information, saving
the
selected subset of biomolecular sequences to a user file, downloading all
listed
biomolecular sequences to a database spreadsheet without classification
information, and displaying classification information on a selected subset of
biomolecular sequences.
If the user chooses to display classification information on a selected
subset of biomolecular sequences, a second GUI may be presented to the user.
In
one embodiment, the second GUI may contain a listing of one or more external
databases used to create the expression profile databases as described above.
Furthermore, for each external database, the GUI may display a list of one or
more fields associated with each external database. In yet another embodiment,
the GUI may allow the user to select or deselect each of the one or more
fields
displayed in the second GU1. In yet another embodiment, the GUI may allow the
user to select or deselect each of the one or more external databases.
The methods of the present application futher relate to the commercial
and other uses of the compositions and methodologies of the present invention.
In one aspect, the methods include the marketing, sale, or licensing of the
compositions and methodologies of the present invention in the context of
providing consumers, i.e., patients, medical practitioners, medical service
providers, researchers, and pharmaceutical. distributors and manufacturers,
with
expression profile databases including, in particular, databases produced in
accordance with the use of the compounds of the present invention.
In another embodiment, the methods of the present invention include
establishing a distribution system for distributing the pharmaceutical
compositions of the present invention for sale, and may optionally include
establishing a sales group for marketing the pharmaceutical composition. Yet
another aspect of the present invention provides a method of conducting target
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discovery comprising identifying, by one or more of the above drug discovery
methods, a test compound, as described above, which modulates the level of
expression of a gene or the activity of a gene product such as perlecan;
conducting therapeutic profiling of agents identified, or further analogs
thereof,
for efficacy and toxicity in animals; and optionally formulating a
pharmaceutical
composition including one or more of the agents identified as having an
acceptable therapeutic profile; and optionally licensing or selling, the
rights for
further drug development of said identified agents.
Pharmaceutical Compositions
In addition to the compounds disclosed herein, the pharmaceutical
compositions of the present invention can further comprise at least one of any
suitable auxiliary such as, but not limited to, diluent, binder, stabilizer,
buffers,
salts, lipophilic solvents, preservative, adjuvant or the like.
Pharmaceutically
I S acceptable auxiliaries are preferred. Examples and methods of preparing
such
sterile solutions are well known in the art and can be found in well known
texts
such as, but not limited to, REMINGTON'S PHARMACEUTICAL SCIENCES (Gennaro,
Ed., 18th Edition, Mack Publishing Co. (1990)). Pharmaceutically acceptable
carriers can be routinely selected that are suitable for the mode of
administration,
solubility and/or stability of the compound.
Pharmaceutical excipients and additives useful in the present invention
include, but are not limited to, proteins, peptides, amino acids, lipids, and
carbohydrates (e.g., sugars, including monosaccharides, di-, tri-, tetra-, and
oligosaccharides; derivatized . sugars such as alditols, aldonic acids,
.esterified
sugars and the like; and polysaccharides or sugar polymers), which can be
present
singly or in combination, comprising alone or in combination in ranges of 1-
99.99% by weight or volume. Exemplary protein excipients include serum
albumin such as human serum albumin (HSA), recombinant human albumin
(rHA), gelatin, casein, and the like. Representative amino acid components,
which can also fimction in a buffering capacity, include alanine, glycine,
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arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine,
leucine,
isoleucine, valine, methionine, phenylalanine, aspartame, and the like.
Carbohydrate excipients suitable for use in the present invention include,
for example, monosaccharides such as fructose, maltose, galactose, glucose, D
mannose, sorbose, and the like; disaccharides, such as lactose, sucrose,
trehalose,
cellobiose, and the like; polysaccharides, such as raffinose, melezitose,
maltodextrins, dextrans, starches, and the like; and alditols, such as
mannitol,
xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), myoinositol and the
like.
The pharmaceutical compositions comprising the compounds of the
present invention can also include a buffer or a pH adjusting agent.
Typically,
the buffer is a salt prepared from an organic acid or base. Representative
buffers
include organic acid salts such as salts of citric acid, ascorbic acid,
gluconic acid,
carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid;
Tris,
tromethamine hydrochloride, or phosphate buffers.
Additionally, pharmaceutical compositions of the invention can include
polymeric excipients/additives such as polyvinylpyrrolidones, ficolls (a
polymeric sugar), dextrates (e.g., cyclodextrins, such as 2-hydroxypropyl-(3-
cyclodextrin), polyethylene glycols, flavoring agents, anti-microbial agents,
sweeteners, antioxidants, anti-static agents, surfactants (e.g., polysorbates
such as
"TWEEN 20" and "TWEEN 80"), lipids (e.g., phospholipids, fatty acids),
steroids (e.g., cholesterol), and chelating agents (e.g., EDTA). These and
additional known pharmaceutical excipients and/or additives suitable for use
in
the present invention are known in the art, e.g., as listed in REMINGTON: THE
SCIENCE & PRACTICE OF PHARMACY (19th ed., WilIiams ~ Williams (1995)) arid
PHYSICIAN'S DESK REFERENCE (52°d ed., Medical Economics (1998)),
the
disclosures of which are expressly entirely incorporated herein by reference.
Pharmaceutical Compositions for Oral Administration
For oral administration in the form of a tablet or capsule, a compound
may be combined with an oral, non-toxic pharmaceutically acceptable inert
carrier such as ethanol, glycerol, water and the like. Moreover, when desired
or
necessary, suitable binders, lubricants, disintegrating agents, and coloring
agents
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may also be incorporated into the mixture. Suitable binders include, without
limitation, starch; gelatin; natural sugars such as glucose or beta-lactose;
corn
sweeteners; natural and synthetic gums such as acacia, tragacanth, or sodium
alginate, carboxymethylcellulose; polyethylene glycol; waxes and the like.
Lubricants used in these dosage forms include, without limitation, sodium
oleate,
sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium
chloride and the like. Disintegrators include, without limitation, starch,
methyl
cellulose, agar, bentonite, xanthan gum and the like.
Formulations of the present invention suitable for oral administration may
be presented as discrete units such as capsules, cachets or tablets each
containing
a predetermined amount of the active ingredient; as a powder or granules; as a
solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as
an
oil-in-water liquid emulsion or a water-in-oil emulsion and as a bolus, etc.
A tablet rnay be made by compression or molding, 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, preservative, surface active or dispersing agent. Molded tablets may
be
made by molding, in a suitable machine, a mixture of the powdered compound
moistened with an inert liquid diluent. The tablets may be optionally coated
or
scored and may be formulated so as to provide a slow or controlled release of
the
active ingredient therein.
In addition, the combinations may be incorporated into biodegradable
polymers allowing for sustained release of the compound, the polymers being
implanted in the vicinity of where drug delivery is desired, for example, at
the
site of restenosis. The biodegradable polymers and their uses are described,
for
example, in detail in Brem et al., 74 J. NEUROSURG. 441-46 (1991). Suitable
examples of sustained-release compositions include semipermeable matrices of
solid hydrophobic polymers containing a compound of the present invention,
which matrices are in the form of shaped articles, e.g., films, or
microcapsules.
Examples of sustained-release matrices include polyesters, hydrogels (for
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example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides
(U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and y ethyl-L-
glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-
glycolic
acid copolymers such as the LUPRON DEPOT~ (Tap Pharmaceuticals, Inc.,
Chicago, IL) (injectable microspheres composed of lactic acid glycolic acid
copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.
Pharmaceutical Compositions for Parenteral Administration
Formulations suitable for parenteral administration include aqueous and
non-aqueous sterile injection solutions which may contain anti-oxidants,
buffers,
bacteriostats and solutes that render the formulation isotonic with the blood
of the
intended recipient; and aqueous and non-aqueous sterile suspensions which may
include suspending agents and thickening agents. The formulations may be
presented in unit-dose or multi-dose containers, for example, sealed ampules
and
vials, and may be stored in a freeze-dried (lyophilized) condition requiring
only
the addition of the sterile liquid carrier, for example, water for injections,
immediately prior to use. Extemporaneous injection solutions and suspensions
may be prepared from sterile powders, granules and tablets ofthe kind
previously
described.
For parenteral administration, sterile suspensions and solutions are
desired.' Isotonic preparations which generally contain suitable preservatives
are
employed when intravenous administration is desired. The pharmaceutical
compositions may be administered parenterally via injection of a formulation
consisting of the active ingredient dissolved in an inert liquid carrier. The
term
"parenteral," as used herein, includes, but is not limited to, subcutaneous
injections, intravenous, intramuscular, intraperitoneal injections, or
infusion
techniques. Acceptable liquid carriers include, for example, vegetable oils
such
as peanut oil, cotton seed oil, sesame oil and the like, as well as organic
solvents
such as solketal, glycerol formal and the like. The formulations may be
prepared
by dissolving or suspending the active ingredient in the liquid carrier such
that
the final formulation contains from about 0.005% to 30% by weight of the
active
ingredient, i.e., a compound of the present invention.
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Pharmaceutical Compositions for Other Routes of Administration
Formulations suitable for topical administration in the mouth include
lozenges comprising the ingredients in a flavored basis, usually sucrose and
acacia or tragacanth; pastilles comprising the active ingredient in an inert
basis
such as gelatin and glycerin, or sucrose and acacia; and mouthwashes
comprising
the compound to be administered in a suitable liquid carrier. The liquid forms
may include suitably flavored suspending or dispersing agents such as the
synthetic and natural gums, for example, tragacanth, acacia, methyl-cellulose
and
the like.
Formulations for rectal administration may be presented as a suppository
with a suitable base comprising, for example, cocoa butter or a salicylate.
Formulations suitable for vaginal administration may be presented as
pessaries, tamports, creams, gels, pastes, foams or spray formulations
containing
in addition to the active ingredient such carriers as are known in the art to
be
appropriate.
The compounds may also be entrapped in microcapsules prepared, for
example, by coacervation techniques or by interfacial polymerization, for
example, hydroxymethylcellulose or gelatin-microcapsules and
poly(methyhnethacylate) microcapsules, respectively, in colloidal drug
delivery
systems (for example, liposomes, albumin microspheres, microemulsions, nano-
particles and nanocapsules) or in macroemulsions. REMINGTON'S
PHARMACEUTICAL SCIENCES (A. Osol ed., 16th ed. (1980)).
In a specific embodiment, the compounds disclosed herein are formulated
as liposomes. Liposomes containing a compound of the present invention are
prepared by methods known in the art. See, e.g., U.S. Patent Nos. 5,013,556;
4,485,045; 4,544,545; WO 97/38731; Epstein et al., 82 Pl2oC. NATL. ACAD. SCI.
USA 3688 (1985); and Hwang et al., 77 PROC. NATL. ACRD. SCI. USA 4030
(1980). The compounds of the present invention can also be administered in the
form of liposome delivery systems such as small unilamellar vesicles, large
unilamellar vesicles, and multilamellar vesicles. Liposomes can be formed from
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a variety of phospholipids such as cholesterol, stearylamine or
phophatidylcholines.
Compounds of the present invention may also be delivered by the use of
monoclonal antibodies as individual carriers to which the compound molecules
are coupled. The compounds of the present invention may also be coupled with
soluble polymers as targetable drug carriers. Such polymers can include
polyvinylpyrrolidone, pyran copolymer,
polyhydroxypropylmethacrylamidephenol, polyhydroxyethylaspartamidephenol,
or polyethyl-eneoxidepolylysine substituted with palmitoyl residue.
Pharmaceutically Acceptable Preservatives
The present invention provides stable formulations as well as preserved
solutions and formulations containing a preservative as well as multi-use
preserved formulations suitable for pharmaceutical or veterinary use,
comprising
at least one compound disclosed herein in a pharmaceutically acceptable
formulation. Formulations in accordance with the present invention may
optionally contain at least one known preservative. Preservatives include, but
are
not limited to, phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl
alcohol,
phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol,
magnesium chloride (e.g., hexahydrate), alkylparaben (methyl, ethyl, propyl,
butyl and the like), benzalkonium chloride, benzethonium chloride, sodium
dehydroacetate and thimerosal, or mixtures thereof in an aqueous diluent. Any
suitable concentration or mixture can be used as known in the art, such as
0.001
5%, or any range or value therein. Non-limiting examples include, no
preservative, 0.1-2% m-cresol, 0.1-3% benzyl alcohol, 0.001-0.5% thimerosal,
0.001-2.0% pheno, 0.0005-1.0% alkylparaben(s), and the like.
Other excipients, e.g., isotonicity agents, buffers, antioxidants,
preservative enhancers, can be optionally added to the diluent. An isotonicity
agent such as glycerin, is commonly used at known concentrations. A
physiologically tolerated buffer is preferably added to provide improved pH
control. The formulations can cover a wide range of pHs, such as from about pH
4 to about pH 10, specifically, a range from about pH 5 to about pH 9, and
more
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specifically, a range of about 6.0 to about 8Ø In one aspect, the
formulations of
the present invention have pH between about 6.8 and about 7.8. Suitable
buffers
include phosphate buffers, for example, sodium phosphate and phosphate
buffered saline (PBS).
Other additives, such as a pharmaceutically acceptable solubilizers like
Tween 20 (polyoxyethylene (20) sorbitan monolaurate), Tween 40
(polyoxyethylene (20) sorbitan monopahnitate), Tween 80 (polyoxyethylene (20)
sorbitan monooleate), Pluronic F68 (polyoxyethylene polyoxypropylene block
copolymers), and PEG (polyethylene glycol) or non-ionic surfactants such as
polysorbate 20 or 80 or poloxamer 184 or 188, Pluronic~ polyls, other block co
polymers, and chelators such as EDTA and EGTA can optionally be added to the
pharmaceutical compositions to reduce aggregation. These additives are
particularly useful if a pump or plastic container is used to administer the
pharmacuetical composition. The presence of pharmaceutically acceptable
I S surfactant mitigates the propensity for the composition to aggregate.
During any of the processes for preparation of the compounds of the
present invention, it may be necessary and/or desirable to protect sensitive
or
reactive groups on any of the molecules concerned. This may be achieved by
means of conventional protecting groups, such as those described in PROTECTIVE
GROUPS IN ORGANIC CHEMISTRY (1973); and GREENE AND WUTS, PROTECTIVE
GROUPS IN ORGANIC SYNTHESIS (1991 ). The protecting groups may be removed
at a convenient subsequent stage using methods known from the art.
Routes of Administration
The invention further relates to the administration of at least one
compound disclosed herein by the following routes, including, but not limited
to
oral, parenteral, subcutaneous, intramuscular, intravenous, intrarticular,
intrabronchial, intraabdominal, intracapsular, intracartilaginous,
intracavitary,
intracelial, intracelebellar, intracerebroventricular, intracolic,
intracervical,
intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic,
intrapericardiac, intraperitoneal, intrapleural, intraprostatic,
intrapulmonary,
intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial,
intrathoracic,
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intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual,
intranasal,
iontophoretic means, or transdermal means.
Pulmonary/Nasal Administration
There are a several desirable features of an inhalation device for
administering a compound of the present invention. For example, delivery by
the
inhalation device is reliable, reproducible, and accurate. For pulmonary
administration, at least one pharmaceutical composition is delivered in a
particle
size effective for reaching the lower airways of the lung or sinuses. The
inhalation device can optionally deliver small dry particles, e.g. less than
about
10 Eun, preferably about 1-5 l.un, for good respirability.
According to the invention, at least one pharmaceutical composition can
be delivered by any of a variety of inhalation or nasal devices known in the
art
for administration of a therapeutic agent by inhalation. Devices capable of
depositing aerosolized formulations in the sinus cavity or alveoli of a
patient
include metered dose inhalers, nebulizers, dry powder generators, sprayers,
and
the like. Other devices suitable for directing pulmonary or nasal
administration
are also known in the art.
All such devices can be used for the administration of a pharmaceutical
composition in an aerosol. Such aerosols may comprise either solutions (both
aqueous and non aqueous) or solid particles. Metered dose inhalers like the
Ventolin° metered dose inhaler, typically use a propellent gas and
require
actuation during inspiration. See, e.g., WO 98/35888; WO 94/16970. Dry
powder inhalers like Turbuhaler° (Astray, Rotahaler° (Glaxo),
Diskus° (Glaxo),
Spiros° inhaler (Dura), devices marketed by Inhale Therapeutics,
and the
Spinhaler° powder inhaler (Fisons), use breath-actuation of a mixed
powder. See
U.S. Patent Nos. 5,458,135; 4,668,218; WO 97/25086; WO 94/08552; WO
94/06498; and EP 0 237 507, each entirely expressly incorporated herein by
reference. Nebulizers like AERx°, Aradigm, the Ultravent°
nebulizer
(Mallinckrodt), and the Acorn II° nebulizer (Marquest Medical
Products), the
above references entirely expressly incorporated herein by reference, produce
aerosols from solutions, while metered dose inhalers, dry powder inhalers,
etc.
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generate small particle aerosols. These specific examples of commercially
available inhalation devices are intended to be a representative of specific
devices
suitable for the practice of the invention, and are not intended as limiting
the
scope of the invention.
Formulations suitable for nasal administration, wherein the carrier is a
solid, include a coarse powder having a particle size, for example, in the
range of
20 to 500 microns which is administered in the manner in which snuff is
administered, i.e., by rapid inhalation through the nasal passage from a
container
of the powder held close up to the nose. Suitable formulations, wherein the
carrier is a liquid, for administration, as for example, a nasal spray or as
nasal
drops, include aqueous or oily solutions of the active ingredient.
A spray comprising a pharmaceutical composition of the present
invention can be produced by forcing a suspension or solution of a compound
disclosed herein through a nozzle under pressure. The nozzle size and
configuration, the applied pressure, and the liquid feed rate can be chosen to
achieve the desired output and particle size. An electrospray can be produced,
for
example, by an electric field in connection with a capillary or nozzle feed.
Advantageously, particles of at least one compound delivered by a sprayer have
a
particle size in a range of about less than 1 pm to less than about 20 pm.
Pharmaceutical compositions of at least one of the compounds of the .
present invention suitable for use with a sprayer typically include a compound
disclosed herein in an aqueous solution at a concentration of about 0.1 mg to
about 100 mg of a compound disclosed herein per ml of solution or mg/gm, or
any range or value therein, including, but not limited to, 0.01, 0.02, 0.03,
0.04,
0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2., 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, l,
2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28,
29, 30, 40, 45, 50, 60, 70, 80, 90 or l00 mg/ml or mg/gm. The pharmaceutical
composition can include agents such as an excipient, a buffer, an isotonicity
agent, a preservative, a surfactant, or other known agents of pharmaceutical
compositions.
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A pharmaceutical composition of the present invention can be
administered by a nebulizer such as a jet nebulizer or an ultrasonic
nebulizer.
Typically, in a jet nebulizer, a compressed air source is used to create a
high-
velocity air jet through an orifice. As the gas expands beyond the nozzle, a
low-
s pressure region is created, which draws a solution of composition protein
through a capillary tube connected to a liquid reservoir. The liquid stream
from
the capillary tube is sheared into unstable filaments and droplets as it exits
the
tube, creating the aerosol. A range of configurations, flow rates, and baffle
types
can be employed to achieve the desired performance characteristics from a
given
jet nebulizer. In an ultrasonic nebulizer, high-frequency electrical energy is
used
to create vibrational, mechanical energy, typically employing a piezoelectric
transducer. This energy is transmitted to the formulation of composition
protein
either directly or through a coupling fluid, creating an aerosol including the
composition protein. Advantageously, particles of the pharmaceutical
I S composition delivered by a nebulizer have a particle size range of from
about less
than 1 Eun to less than about 20 Eun.
Pharmaceutical compositions comprising a compound of the present
invention suitable for use with a nebulizer, either jet or ultrasonic,
typically
include a concentration of about 0.1 mg to about 100 mg of a compound
disclosed herein per ml of solution or mg/gm, or any range or value therein
including, but not limited to, the individual amounts disclosed for spray
compositions. The pharmaceutical composition can include other pharmaceutical
agents such as an excipient, a buffer, an isotonicity agent, a preservative, a
surfactant, and those known in the art for use in nebulizer administration.
In a metered dose inhaler (MDI), a propellant, a compound of the present
invention, and any excipients or other additives are contained in a cannister
as a
mixture including a liquefied, compressed gas. Actuation of the metering valve
releases the mixture as an aerosol, preferably containing a particle size
range of
from about less than 1 pm to Less than about 20 p.m.
The desired aerosol particle size can be obtained by employing a
formulation of a compound of the present invention produced by various methods
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known to those of skill in the art including, but not limited to, jet-milling,
spray
drying, critical point condensation, and the like. Suitable metered dose
inhalers
include those manufactured by 3M or Glaxo and employing a hydrofluorocarbon
propellant.
Pharmaceutical compositions for use with a metered-dose inhaler device
will generally include a finely divided powder containing a compound disclosed
herein as a suspension in a non-aqueous medium, for example, suspended in a
propellant with the aid of a surfactant. The propellant can be any
conventional
material employed for this purpose such as chlorofluorocarbon, a
hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon including
trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol
and
1,1,1,2-tetrafluoroethane, HFA-134a (hydrofluroalkane-134a), HFA-227
(hydrofluroalleane-227), or the like. In one embodiment, the propellant is a
hydrofluorocarbon. The surfactant can be chosen to stabilize the compound of
the present invention as a suspension in the propellant, to protect the active
agent
against chemical degradation, and the like. Suitable surfactants include
sorbitan
trioleate, Soya lecithin, oleic acid, or the like. In some cases solution
aerosols are
preferred using solvents such as ethanol. One of ordinary skill in the art
will
recognize that the methods of the present invention can be achieved by
pulmonary administration of a compound disclosed herein via devices not
described herein.
For absorption through mucosal surfaces, the compositions and methods
of the present invention for administering a compound disclosed herein include
an emulsion comprising a plurality of submicron particles, a mucoadhesive
macromolecule, a bioactive peptide, and an aqueous continuous phase, which
promotes absorption through mucosal surfaces by achieving mucoadhesion of the
emulsion particles. See, e.g., U.S. Patent No. 5,514,670. Mucous surfaces
suitable for application of the emulsions of the present invention can include
corneal, conjunctival, buccal, sublingual, nasal, vaginal, pulmonary,
abdominal,
intestinal, and rectal routes of administration. Pharmaceutical compositions
for
vaginal or rectal administration such as suppositories, can contain as
excipients,
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for example, polyallcyleneglycols, vaseline, cocoa butter, and the like.
Pharmaceutical composition s for intranasal administration can be solid and
contain excipients, for example, lactose or can be aqueous or oily solutions
of
nasal drops. For buccal administration, excipients include sugars, calcium
stearate, magnesium stearate, pregelinatined starch, and the like. See, e.g.,
U.S.
Patent No. 5,849,695.
1n another embodiment, the pharmaceutical compositions of the present
invention may be administered via transdermal routes using forms of
transdermal
skin patches well known to those of ordinary skill in that art. For
transdermal
administration, a compound of the present invention is encapsulated in a
delivery
device such as a liposome or polymeric nanoparticles, microparticle,
microcapsule, or microspheres (referred to collectively as microparticles
unless
otherwise stated). A number of suitable devices are known, including
microparticles made of synthetic polymers such as polyhydroxy acids such as
polylactic acid, polyglycolic acid and copolymers thereof, polyorthoesters,
polyanhydrides, and polyphosphazenes, and natural polymers such as collagen,
polyamino acids, albumin and other proteins, alginate and other
polysaccharides,
and combinations thereof. See, e.g., U.S. Patent No. 5,814,599. To be
administered in the form of a transdermal delivery system, the dosage
administration may be, for example, continuous rather than intermittent
throughout the dosage regimen.
Formulations suitable for topical administration to the skin may be
presented as ointments, creams, gels and pastes comprising the ingredient to
be
administered in a pharmaceutical acceptable carrier. A preferred topical
delivery
system is a transdermal patch comprising a compound of the present invention.
Topical compositions containing a compound of the present invention
may be admixed with a variety of carrier materials well known in the art
including alcohols, aloe vera gel, allantoin, glycerine, vitamin A and E oils,
mineral oil, PPG2 myristyl propionate and the like to form, for example,
alcoholic solutions, topical cleansers, cleansing creams, skin gels, skin
lotions,
and shampoos in cream or gel formulations. Examples of such carriers and
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methods of formulation may be found in REMINGTON'S PHARMACEUTICAL
SCIENCES (1990). Pharmaceutical formulations may contain from about 0.005%
to about 10% by weight of the active ingredient. In one embodiment, the
pharmaceutical formulations contain from about 0.01% to 5% by weight of the
compound of the present invention.
It can be sometimes desirable to deliver the compounds of the present
invention to the subject over prolonged periods of time, for example, for
periods
of one week to one year from a single administration. Certain medical devices
may be employed to provide a continuous intermittent or on demand dosing of a
patient. The devices may be a pump of diffusion apparatus, or other device
containing a reservoir of drug and optionally diagnostic or monitoring
components to regulate the delivery of the drug. Various slow-release, depot
or
implant dosage forms can be utilized. For example, a dosage form can contain a
pharmaceutically acceptable non-toxic salt of compound disclosed herein that
has
a low degree of solubility in body fluids, for example, (a) an acid addition
salt
with a polybasic acid such as phosphoric acid, sulfuric acid, citric acid,
tartaric
acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalene
mono- or di-sulfonic acids, polygalacturonic acid, and the like; (b) a salt
with a
polyvalent metal cation such as zinc, calcium, bismuth, barium, magnesium,
aluminum, copper, cobalt, nickel, cadmium and the like, or with an organic
cation
formed from e.g., N,N'-dibenzyl-ethylenediamine or ethylenediamine; or (c)
combinations of (a) and (b) e.g., a zinc tannate salt. Additionally, the
compounds
of the present invention or, preferably, a relatively insoluble salt such as
those
just described, can be formulated in a gel, for example, an aluminum
monostearate gel with, e.g., sesame oil, suitable for injection. Exemplary
salts
include, but are not limited to, zinc salts, zinc tannate salts, pamoate
salts, and the
like. Another type of slow-release depot formulation for injection would
contain
the compound or salt dispersed or encapsulated in a slow degrading, non-toxic,
non-antigenic polymer such as a polylactic acid/polyglycolic acid polymer, for
example, as described in U.S. Patent No. 3,773,919. The compounds or
relatively insoluble salts thereof such as those described above can also be
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formulated in cholesterol matrix silastic pellets, particularly for use in
animals.
Additional slow-release, depot or implant formulations, e.g., gas or liquid
liposomes are known in the literature. See, e.g., U.S. Patent No. 5,770,222;
SUSTAINED AND CONTROLLED RELEASE DRUG DELIVERY SYSTEMS (1978.
Other examples include provision of the compounds of the present
invention to be administered by sustained release delivery system containing a
biodegradable composition. The biodegradable composition may be composed
of a biodegradable, water-coagulable, non-polymeric material and a
biocompatible, non-toxic organic solvent that is miscible to dispersible in an
aqueous medium. The delivery system may be implanted at an implant site
causing the solvent to dissipate, disperse or leach from the composition into
surrounding tissue fluid through a resulting microporous matrix.
As used herein, the term "implant site" is meant to include a site, in or on
which the non-polymeric composition is applied. Implantation or implant site
can also include the incorporation of the pharmaceutical composition
comprising
at least one compound of the present invention with a solid device. For
example,
the pharmaceutical composition is incorporated into a coating on a stmt that
is
implanted into a subject. Additionally, other solid or biodegradeable
materials
can be used as a substrate on which the pharmaceutical composition is applied.
The coated material, comprising the pharmaceutical composition is then
implanted, inserted or is adjacent to the subject or patient. The term
"biodegradable" means that the non-polymeric material andlor matrix of the
implant will degrade over time by the action of enzymes, by simple or
enzymatically. catalyzed hydrolytic action andlor by other similar mechanisms
in
the human body. By "bioerodible," it is meant that the implant matrix will
erode
or degrade over time due, at feast in part, to contact with substances found
in the
surrounding tissue fluids, cellular action, and the like. By "bioabsorbable,"
it is
meant that the non-polymeric matrix will be broken down and absorbed within
the human body, for example, by a cell, a tissue, and the like.
Non-polymeric materials that can be used in the composition generally
are those that are biocompatible, substantially insoluble in water and body
fluids,
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and biodegradable and/or bioerodible. The non-polymeric material is capable of
being at least partially solubilized in a water-soluble organic solvent. The
non-
polymeric materials are also capable of coagulating or solidifying to form a
solid
implant matrix. The non-polymeric material is combined with a compatible and
suitable organic solvent to form a composition that has the desired
consistency
ranging from watery to viscous to a spreadable putty or paste.
Suitable organic solvents are those that are biocompatible,
pharmaceutically-acceptable, and will at least partially dissolve the non-
polymeric material. The organic solvent has a solubility in water ranging from
miscible to dispersible. Optionally, a pore-forming agent can be included in
the composition to generate additional pores in the implant matrix. The pore
forming agent can be any organic or inorganic, pharmaceutically-acceptable
substance that is substantially soluble in water or body fluid, and will
dissipate
from the coagulating non-polymeric material and/or the solid matrix of the
implant into surrounding body fluid at the implant site.
The compounds of the present invention are capable of providing a local
or systemic biological, physiological or therapeutic effect in the body of an
animal. In formulating some pharmaceutical compositions described herein, the
compound is preferably soluble or dispersible in the non-polymeric composition
to form a homogeneous mixture, and upon implantation, becomes incorporated
into the implant matrix. As the solid matrix degrades over time, the compound
is
capable of being released from the matrix into the adjacent tissue fluid, and
to the
pertinent body tissue or organ, either adjacent to or distant from the implant
site,
preferably at a controlled rate. The release. of the compound from the matrix
may
be varied, for example, by the solubility of the compound in an aqueous
medium,
the distribution of the compound within the matrix, the size, shape, porosity,
and
solubility and biodegradability of the solid matrix. See e.g. U.S. Patent No.
5,888,533. The amounts and concentrations of ingredients in the composition
administered to the patient will generally be effective to accomplish the task
intended.
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Compounds of the present invention may be administered by bioactive
agent delivery systems containing microparticles suspended in a polymer
matrix.
The microparticles may be microcapsules, microspheres or nanospheres currently
lenown in the art. The microparticles should be capable of being entrained
intact
within a polymer that is or becomes a gel once inside a biological
environment.
The microparticles can be biodegradable or non-biodegradable. Many
microencapsulation techniques used to incorporate a bioactive agent into a
microparticle carrier are taught in the art. See e.g. U.S. Patent Nos.
4,652,441;
S,I00,669; 4,438,253; and 5,665,428.
A preferred polymeric matrix will be biodegradable and exhibit water
solubility at low temperature and will undergo reversible thermal gelation at
physiological mammalian body temperatures. The polymeric matrix is capable of
releasing the substance entrained within its matrix over time and in a
controlled
manner. The polymers are gradually degraded by enzymatic or non-enzymatic
hydrolysis in aqueous or physiological environments. See e.g. U.S. Patent No.
6,287,588.
Compounds of the present invention may be administered by a drug
delivery composition comprising microparticles containing at least one
chemotherapeutic agent and at least one chemosensitizer suspended in a polymer
matrix. The microparticles may be microcapsules, microspheres or nanospheres
currently known in the art. The microparticles should be biodegradable and
stable in physiological environments. The microparticles also permit diffusion
of
the chemotherapeutic agent and chemosensitizer from the core through the
matrix
at a predetermined release rate. Ionic chemotherapeutic agents are suitable
for
use in the delivery composition of the invention. Ionic chemosensitizers are
suitable for use in the delivery composition of the invention. The drug
delivery
compositions may be delivered to a target site through a variety of known
routes
of administration. Dosages of the chemotherapeutic agent and chemosensitiThe
drug delivery compositions may be delivered to a target site through a variety
of
known routes of administration. Dosages of the chemotherapeutic agent and
chemosensitizer incorporated in the drug delivery composition will depend on
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individual needs, the desired effect and on the chosen route of
administration.
See e.g. WO 98/50018.
Dosage Determinations
In general, the compounds disclosed herein may be used alone or in
concert with other therapeutic agents at appropriate dosages defined by
routine
testing in order to obtain optimal efficacy while minimizing any potential
toxicity. The dosage regimen utilizing a compound of the present invention may
be selected in accordance with a variety of factors including type, species,
age,
weight, sex, medical condition of the patient; the severity of the condition
to be
treated; the route of administration; the renal and hepatic function of the
patient;
and the particular compound employed. A physician or veterinarian of ordinary
skill can readily determine and prescribe the effective amount of the drug
required to prevent, counter, or arrest the progress of the condition.
Optimal precision in achieving concentrations of drug within the range
that yields maximum efficacy with minimal toxicity may require a regimen based
on the kinetics of the compound's availability to one or more target sites.
Distribution, equilibrium, and elimination of a drug may be considered when
determining the optimal concentration for a treatment regimen. The dosages of
a
compound disclosed herein may be adjusted when combined to achieve desired
effects. On the other hand, dosages of these various therapeutic agents may be
independently optimized and combined to achieve a synergistic result wherein
the pathology is reduced more than it would be if either agent were used
alone.
In particular, toxicity and therapeutic efficacy of a compound disclosed
herein may be determined by standard pharmaceutical procedures in cell
cultures
or experimental animals, e.g., for determining the LDso (the dose lethal to
50% of
the population) and the EDso (the dose therapeutically effective in 50% of the
population). The dose ratio between toxic and therapeutic effect is the
therapeutic index and it may be expressed as the ratio LDso/EDso. Compounds
exhibiting large therapeutic indices are preferred except when cytotoxicity of
the
compound is the activity or therapeutic outcome that is desired. Although
compounds that exhibit toxic side effects may be used, a delivery system can
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target such compounds to the site of affected tissue in order to minimize
potential
damage to uninfected cells and, thereby, reduce side effects. Generally, the
compounds of the present invention may be administered in a manner that
maximizes efficacy and minimizes toxicity.
Data obtained from cell culture assays and animal studies may be used in
formulating a range of dosages for use in humans. The dosages of such
compounds lies preferably within a range of circulating concentrations that
include the EDSO with little or no toxicity. The dosage may vary within this
range
depending upon the dosage form employed and the route of administration
utilized. For any compound used in the methods of the invention, the
therapeutically effective dose may be estimated initially from cell culture
assays.
A dose may be formulated in animal models to achieve a circulating plasma
concentration range that includes the ICSO (the concentration of the test
compound
that achieves a half maximal inhibition of symptoms) as determined in cell
culture. Such information may be used to accurately determine useful doses in
humans. Levels in plasma may be measured, for example, by high performance
liquid chromatography.
Moreover, the dosage administration of the pharmaceutical compositions
of the present invention may be optimized using a
pharmacokinetic/pharmacodynamic modeling system. For example, one or more
dosage regimens may be chosen and a pharmacokinetic/pharmacodynamic model
may be used to determine the pharmacokinetic/pharmacodynamic profile of one
or more dosage regimens. Next, one of the dosage regimens for administration
may be selected which achieves the desired pharmacokinetic/pharmacodynamic
response based on the particular pharmacokineticlpharmacodynamic profile. See
WO 00/67776, which is entirely expressly incorporated herein by reference.
Methods are lenown in the art for determining effective doses for
therapeutic and prophylactic purposes for the disclosed pharmaceutical
compositions or the disclosed drug combinations, whether or not formulated in
the same composition. For therapeutic purposes, the term "jointly effective
amount," as used herein, means that amount of each active compound or
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pharmaceutical agent, alone or in combination, that elicits the biological or
medicinal response in a tissue system, animal or human that is being sought by
a
researcher, veterinarian, medical doctor or other clinician, which includes
alleviation of the symptoms of the disease or disorder being treated. For
prophylactic purposes (i.e., inhibiting the onset or progression of a
disorder), the
term "jointly effective amount" refers to that amount of each active compound
or
pharmaceutical agent, alone or in combination, that inhibits in a subject the
onset
or progression of a disorder as being sought by a researcher, veterinarian,
medical
doctor or other clinician. Thus, the present invention provides combinations
of
two or more therapeutic agents wherein, for example, (a) each therapeutic
agent
is administered in an independently therapeutically or prophylactically
effective
amount; (b) at least one therapeutic agent in the combination is administered
in
an amount that is sub-therapeutic or subprophylactic if administered alone,
but is
therapeutic or prophylactic when administered in combination with the second
or
additional therapeutic agents according to the invention; or (c) both
therapeutic
agents are administered in an amount that is subtherapeutic or sub-
prophylactic if
administered alone, but are therapeutic or prophylactic when administered
together. Combinations of three or more therapeutic agents are analogously
possible. Methods of combination therapy include coadministration of a single
formulation containing all active agents; essentially contemporaneous
administration of more than one formulation; and administration of two or more
active agents separately formulated.
Dosages
More specifically, the pharmaceutical compositions may be administered
in a single daily dose, or the total daily dosage may be administered in
divided
doses of two, three, or four times daily. In the case of oral administration,
the
daily dosage of the compositions may be varied over a wide range from about
0.0001 to about 1,000 mg per patient, per day. The range may more particularly
be from about 0.001 mg/Icg to 10 mg/kg of body weight per day, about 0.1-100
mg, about I .0-50 mg or about 1.0-20 mg per day for adults (at about 60 kg).
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The daily dosage of the pharmaceutical compositions may be varied over
a wide range from about 0.01 to about 1000 mg per adult human per day. For
oral administration, the pharmaceutical compositions are preferably provided
in
the form of tablets containing from about 0.1 mg to about 1000 mg of the
compound or 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0, 15.0, 100, 150, 200, 250, 300,
350,
400, 450, 500, 550, 600, 650, 700, 800, 900, or 1000 milligrams of the active
compound for the symptomatic adjustment of the dosage to the patient to be
treated. An effective amount of the drug is ordinarily supplied at a dosage
level
of from about 0.1 mg/kg to about 20 mg/kg of body weight per day. In one
embodiment, the range is from about 0.2 mg/kg to about 10 mg/kg of body
weight per day. In another embodiment, the range is from about 0.5 mg/kg to
about 10 mg/kg of body weight per day. The compounds may be administered on
a regimen of about 1 to about 10 times per day.
In the case of injections, it is usually convenient to give by an intravenous
I 5 route in an amount of about 0.01-30 mg, about 0.1-20 mg or about 0.1-10 mg
per
day to adults (at about 60 kg). In the case of other animals, the dose
calculated
for 60 kg may be administered as well.
Doses of a compound of the present invention can optionally include
0.0001 to 1,000 mg/kg/administration, or 0.001 to 100.0 mg/kg/administration,
from 0.01 to 10 mg/kg/administration, from 0.1 to 10 mg/kg/administration,
including0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9,1,2,3,4,5,6,7,8,9,10,11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33,
34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,
53, 54, 55,
56, 57, 58, 59, 60, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,
76, 77, 78,
79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,
98, 99
and/or 100-500 mg/kg/administration or any range, value or fraction thereof,
or to
achieve a serum concentration of 0.1, 0.5, 0.9, 1.0, 1.1, 1.2, 1.5, 1.9, 2.0,
2.5, 2.9,
3.0,3.5,3.9,4.0,4.5,4.9,5.0,5.5,5.9,6.0,6.5,6.9,7.0,7.5,7.9,8.0,8,5,8.9,
9.0, 9.5, 9.9, 10, 10.5, 10.9, 11, 11.5, 11.9, 20, 12.5, 12.9, 13.0, 13.5,
13.9, 14.0,
3 0 1 4.5, 4.9, 5.0, 5.5., 5.9, 6.0, 6.5, 6.9, 7.0, 7.5, 7.9, 8.0, 8.5, 8.9,
9.0, 9.5, 9.9, 1 0,
10.5,10.9,11,11.5,11.9,12,12.5,12.9,13.0,13.5,13.9,14,14.5,15,15.5,15.9,
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16, 16.5, 16.9, 17, 17.5, 17.9, 18, 18.5, 18.9, 19, 19.5, 19.9, 20, 20.5,
20.9, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,
90, 96,
100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000,
3500,
4000, 4500, and/or 5000 yghnl serum concentration per single or multiple
administration or any range, value or fraction thereof.
As a non-limiting example, treatment of humans or animals can be
provided as a one-time or periodic dosage of a compound of the present
invention
0.1 to 100 mg/kg such as 0.5, 0.9, I .0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45,
50, 60, 70,
80, 90 or 100 mg/kg, per day, on at least one of day l, 2, 3, 4, 5, 6, 7, 8,
9, 10, 1 l,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33,
34, 35, 36, 37, 38, 39, or 40, or alternatively or additionally, at least one
of week
l, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, I5, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
45, 46, 47,
48, 49, 50, 51, or 52, or alternatively or additionally, at least one of 1, 2,
3, 4, 5,
6" 7, 8, 9, 10, I 1, 12, 13, 14, 15, 16, 17, 18, 19, or 20 years, or any
combination
thereof, using single, infusion or repeated doses.
Specifically, the pharmaceutical compositions of the present invention
may be administered at least once a week over the course of several weeks. In
one embodiment, the pharmaceutical compositions are administered at least once
a week over several weeks to several months. In another embodiment, the
pharmaceutical compositions are administered once a week over four to eight
weeks. In yet another embodiment, the pharmaceutical compositions are
administered once a week over four weeks.
More specifically, the pharmaceutical compositions may be administered
at least once a day for about 2 days, at least once a day for about 3 days, at
least
once a day for about 4 days, at least once a day for about 5 days, at least
once a
day for about 6 days, at least once a day for about 7 days, at least once a
day for
about 8 days, at least once a day for about 9 days, at least once a day for
about 10
days, at least once a day for about I 1 days , at least once a day for about
12 days ,
at least once a day for about 13 days , at least once a day for about 14 days,
at
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least once a day for about l 5 days, at least once a day for about 16 days, at
least
once a day for about 17 days, at least once a day for about 18 days, at least
once a
day for about 19 days, at least once a day for about 20 days, at least once a
day
for about 21 days, at least once a day for about 22 days, at least once a day
for
about 23 days, at least once a day for about 24 days, at least once a day for
about
25 days, at least once a day for about 26 days, at least once a day for about
27
days, at least once a day for about 28 days, at least once a day for about 29
days,
at least once a day for about 30 days, or at least once a day for about 31
days.
Alternatively, the pharmaceutical compositions may be administered
about once every day, about once every 2 days, about once every 3 days, about
once every 4 days, about once every 5 days, about once every 6 days, about
once
every 7 days, about once every 8 days, about once every 9 days, about once
every
10 days, about once every I 1 days, about once every 12 days, about once every
l3 days, about once every 14 days, about once every 15 days, about once every
I S 16 days, about once every 17 days, about once every 18 days, about once
every
19 days, about once every 20 days, about once every 21 days, about once every
22 days, about once every 23 days, about once every 24 days, about once every
25 days, about once every 26 days, about once every 27 days, about once every
28 days, about once every 29 days, about once every 30 days, or about once
every 31 days.
The pharmaceutical compositions of the present invention may
alternatively be administered about once every week, about once every 2 weeks,
about once every 3 weeles, about once every 4 weeks, about once every 5 weeks,
about once every 6 weeks, about once every 7 weeks, about once every 8 weeks,
about once every 9 weeks, about once every 10 weeks, about once every 11
weeks, about once every 12 weeks, about once every 13 weeks, about once every
14 weeks, about once every 15 weeks, about once every 16 weeks, about once
every 17 weeks, about once every 18 weeks, about once every 19 weeks, about
once every 20 weeks.
Alternatively, the pharmaceutical compositions of the present invention
may be administered about once every month, about once every 2 months, about
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once every 3 months, about once every 4 months, about once every 5 months,
about once every 6 months, about once every 7 months, about once every 8
months, about once every 9 months, about once every 10 months, about once
every 1 I months, or about once every 12 months.
Alternatively, the pharmaceutical compositions may be administered at
least once a week for about 2 weelcs, at least once a week for about 3 weeks,
at
least once a week for about 4 weeks, at least once a week for about 5 weeks,
at
least once a week for about 6 weeks, at least once a week for about 7 weeks,
at
least once a week for about 8 weeks, at least once a week for about 9 weeks,
at
least once a week for about 10 weeks, at least once a week for about 11 weeks,
at
least once a week for about 12 weeks, at least once a week for about 13 weeks,
at
least once a week for about 14 weeks, at least once a week for about 15 weeks,
at
least once a week for about 16 weeks, at least once a week for about 17 weeks,
at
least once a week for about 18 weeks, at least once a week for about 19 weeks,
or
at least once a week for about 20 weeks.
Alternatively the pharmaceutical compositions may be administered at
least once a week for about 1 month, at least once a week for about 2 months,
at
least once a week for about 3 months, at least once a week for about 4 months,
at
least once a week for about 5 months, at least once a week for about 6 months,
at
least once a week for about 7 months, at least once a week for about 8 months,
at
least once a week for about 9 months, at least once a week for about 10
months,
at least once a week for about 11 months, or at least once a week for about 12
months.
Combination Therapy
In addition, co-administration or sequential administration of the
compounds of the present invention and other therapeutic agents may be
desirable, such as chemotherapeutic agents, immunosuppressive agents,
cytokines, cytotoxic agents, nucleolytic compounds, radioactive isotopes,
receptors, and pro-drug activating enzymes, which may be naturally occurring
or
produced by recombinant methods. The combined administration includes co-
administration, using separate formulations or a single pharmaceutical
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formulation, and consecutive administration in either order, wherein
preferably
there is a time period while both (or all) active therapeutic agents
simultaneously
exert their biological activities.
The compounds of this invention may be administered in combination
with at least one selected from the group consisting of an antirheumatic
(e.g.,
methotrexate, auranofin, aurothioglucose, azathioprine, etanercept, gold
sodium
thiomalate, hydroxychloroquine sulfate, leflunomide, sulfasalzine), a muscle
relaxant, a narcotic, a non-steroid anti-inflammatory drug (NSAID), an
analgesic,
an anesthetic, a sedative, a local anesthetic, a neuromuscular blocker, an
anti
cancer, an antimicrobial (e.g., aminoglycoside, an antifungal, an
antiparasitic, an
antiviral, a carbapenem, cephalosporin, a flurorquinolone, a macrolide, a
penicillin, a sulfonamide, a tetracycline, another antimicrobial), an anti-
psoriatic,
a corticosteriod, an anabolic steroid, a diabetes-related agent, a mineral, a
nutritional, a thyroid agent, a vitamin, a calcium-related hormone, an
IS antidiarrheal, an anti-tussive, an anti-emetic, an anti-ulcer, a laxative,
an
anticoagulant, an erythropieitin (e.g., epoetin alpha), a filgrastim (e.g., G-
CSF,
Neupogen), a sargramostim (GM-CSF, Leukine), an immunization, an
immunoglobulin, an immunosuppressive (e.g., basiliximab, cyclosporine,
daclizumab), a growth hormone, a hormone replacement drug, an estrogen
receptor modulator, a mydriatic, a cycloplegic, an alkylating agent, an anti
metabolite, a mitotic inhibitor, a radiopharmaceutical, an anti-depressant,
anti
manic agent, an anti-psychotic, an anxiolytic, a hypnotic, a sympathomimetic,
a
stimulant, donepezil, tacrine, an asthma medication, a beta agonist, an
inhaled
steroid, a leukotriene inhibitor, a methylxanthine, a cromolyn, an epinephrine
or
analog thereof, dornase alpha (Pulmozyme), or a cytokine.
Such anti-cancer or antimicrobial compounds can also include toxin
molecules that are associated, bound, co-formulated, co-administered or
sequentially administered, in either order, with at least one of the compounds
of
the present invention. The toxin can optionally act to selectively kill the
pathologic cell or tissue. The pathologic cell can be a cancer or other cell.
Such
toxins can be, but are not limited to, purified or recombinant toxin or toxin
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fragment comprising at least one functional cytotoxic domain of toxin, e.g.,
selected from at least one of ricin, diphtheria toxin, a venom toxin, or a
bacterial
toxin. The term toxin also includes both endotoxins and exotoxins produced by
any naturally occurring, mutant or recombinant bacteria or viruses which may
cause any pathological condition in humans and other mammals, including toxin
shock, which can result in death. Such toxins may include, but are not limited
to,
enterotoxigenic E. coli heat-labile enterotoxin (LT), heat-stable enterotoxin
(ST),
Shigella cytotoxin, Aeromonas enterotoxins, toxic shock syndrome toxin-1
(TSST-1), Staphylococcal enterotoxin A (SEA), B (SEB), or C (SEC),
Streptococcal enterotoxins and the like. Such bacteria include, but are not
limited
to, strains of a species of enterotoxigenic E. coli (ETEC), enterohemorrhagic
E.
coli (e.g., strains of serotype 0157:H7), Staphylococcus species (e.g.,
Staphylococcus aureus, Staphylococcus pyogenes), Shigella species (e.g.,
Shigella dysenteriae, Shigella flexneri, Shigella boydii, and Shigella
sonnei),
I S Salmonella species (e.g., Salmonella typhi, Salmonella cholera-suis,
Salmonella
enteritidis), Clostridium species (e.g., Clostridium perfringens, Clostridium
dificile, Clostridium botulinum), Camphlobacter species (e.g., Camphlobacter
jejuni, Camphlobacter fetus), Heliobacter species, (e.g., Heliobacter pylori),
Aeromonas species (e.g., Aeromonas sobria, Aeromonas hydrophila, Aeromonas
caviae), Pleisomonas shigelloides, Yersina enterocolitica, Vibrios species
(e.g.,
Vibrios cholerae, Vibrios parahemolyticus), Klebsiella species, Pseudomonas
aeruginosa, and Streptococci. See, e.g., Stein, ed., INTERNAL MEDICINE, 3rd
ed., pp I-13, Little, Brown and Co., Boston, (1990); Evans et al., eds.,
Bacterial
Infections of Humans: Epidemiology and Control, 2d. Ed., pp 239-254, Plenum
Medical Book Co., New York (1991); Mandell et al, Principles and Practice of
Infectious Diseases, 3d. Ed., Churchill Livingstone, New York (1990); Berkow
et
al, eds., The Merck Manual, 16th edition, Merck and Co., Rahway, N.J., 1992;
Wood et al, FEMS Microbiology Immunology, 76:121-134 (1991); Marrack et al,
Science, 248:705-711 (1990), the contents of which references are incorporated
entirely herein by reference.
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More specifically, the compound of the present invention may be
administered in combination with at least one immunosuppressive agent for use
in, for example, treating or preventing a vascular occlusive conditions such
as
transplant vasculopathy. Suitable immunosuppressive agents include, but are
not
limited to, CellCept (Roche Labs.), Gengraf (Abbott Labs., Inc.), Micrhogam
(Ortho-Clinical), Neoral (Novartis), Orthoclone OKT3 (Ortho-Biotech), Prograf
(Fujisawa), Rapamune (Wyeth-Ayerst), Sandimmune (Novartis), Thymoglobulin
(SangStat), Zenapax (Ruche).
In one embodiment, the therapeutic agent administered simultaneously or
sequentially, in either order and at various times with a compound of the
present
invention, comprises a chemotherapeutic agent. A "chemotherapeutic agent" is a
compound useful in the treatment of cancer. Examples of chemotherapeutic
agents include, but are not limited to, alkylating agents such as thiotepa and
cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and
piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa;
ethylenimines and methylamelamines including altretamine,
triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide
and trimethylolomelamine; nitrogen mustards such as chlorambucil,
chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine,
mechlorethamine oxide hydrochloride, melphalan, novembiehin, phenesterine,
prednimustine, trofosfamide, uracil mustard; nitroureas such as cannustine,
chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics
such as
aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin,
calicheamicin, carabicin, carminomycin, carzinophilin, chromoinycins,
dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,
doxorubicin, epirubicin, esorubicin, idambicin, marcellomycin, mitomycins,
mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin,
puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-
fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate,
pteropterin, trimetrexate; purine analogs such as fludarabine, 6-
mercaptopurine,
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thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine,
6-
azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine,
floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate,
epitiostanol, rnepitiostane, testolactone; anti-adrenals such as
aminoglutethimide,
mitotane, trilostane; folic acid replenisher such as frolinic acid;
aceglatone;
aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil;
bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine;
elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan;
lonidamine;
mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet;
pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSI~~;
razoxane; sizofrran; spirogermanium; tenuazonic acid; triaziquone; 2, 2',2"-
trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine;
mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C");
cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel (TAXOL~, Bristol-Myers
Squibb Oncology, Princeton, NJ) and doxetaxel (TAXOTERE~, Rhone-Poulenc
Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine;
mercaptopurine; methotrexate; platinum analogs such as cisplatin and
carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin
C;
mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide;
daunomycin; aminopterin; xeloda; ibandronate; CPT-I1; topoisomerase inhibitor
RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamicins;
capecitabine; and pharmaceutically acceptable salts, acids or derivatives of
any of
the above. Also included in this definition are anti-hormonal agents that act
to
regulate or inhibit hormone action on tumors such as anti-estrogens including
for
example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4
hydroxytamoxifen, trioxifene, keoxifene, onapristone, and toremifene
(Fareston);
and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide,
and
goserelin; and pharmaceutically acceptable salts, acids or derivatives of any
of
the above.
In another embodiment, the therapeutic agent comprises a cytokine. The
term "cytokine" is a generic term for proteins released by one cell population
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which act on another cell as intercellular mediators. Examples of such
cytokines
are lymphokines, monokines, and traditional polypeptide hormones. Included
among the cytokines are growth hormones such as human growth hormone, N-
methionyl human growth hormone, and bovine growth hormone; parathyroid
hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein
hormones such as follicle stimulating hormone (FSH), thyroid stimulating
hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast
growth factor; prolactin; placental lactogen; tumor necrosis factor-a and -(3;
mullerian-inhibiting substance; mouse gonadotropin-associated peptide;
inhibin;
activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO);
nerve growth factors such as NGF-(3; platelet growth factor; transforming
growth
factors (TGFs) such as TGF-a and TGF-(3; insulin-like growth factor-I and -II;
erythropoietin (EPO); osteoinductive factors; interferons such as interferon-
a, -(3
and -y; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);
I S granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (GCSF);
interleukins (ILs) such as IL-1, IL-la, IL-2, IL-3, IL-4, IL-S, IL-6, IL-7, IL-
8, IL-
9, IL-I I, IL-12, IL-I5; a tumor necrosis factor such as TNF-a or TNF-(i; and
other polypeptide factors including LIF and kit ligand (ILL). As used herein,
the
teen cytoleine includes proteins from natural sources or from recombinant cell
culture and biologically active equivalents of the native sequence cytokines.
In another embodiment, the compounds of the present invention may be
administered in combination with an anti-inflammatory agent including, but not
limited to, adrenocortical steroids (cortisol, cortisone, fludrocortisone,
prednisone, prednisolone, 6a-methylprednisolone, triamcinolone, betamethasone,
and dexamethasone), non-steroidal agents (salicylic acid derivatives, i.e.,
aspirin;
para-aminophenol derivatives, i.e., acetominophen; indole and indene acetic
acids
(indomethacin, sulindac, and etodalac), heteroaryl acetic acids (tolmetin,
diclofenac, and ketorolac), arylpropionic acids (ibuprofen and derivatives),
anthranilic acids (mefenamic acid, and meclofenamic acid), enolic acids
(piroxicam, tenoxicam, phenylbutazone, and oxyphenthatrazone), nabumetone,
gold compounds (auranofin, aurothioglucose, gold sodium thiomalate).
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Commercially available nonsteroidal anti-inflammatory drugs include, but are
not
limited to, Anaprox (Roche Labs.), Arthrotec (Searle), Cataflam (Novartis),
Celebrex (Pfizer), , Clinoril (Merck), Dolobid (Merck), Feldene (Pfizer),
Indocin
(Mercle), Lodine (Wyeth-Ayerst), Mobic (Boehringer Ingelheim), Motrin
(McNeil Consumer), Naprosyn (Roche Labs.), Orudis (Wyeth-Ayerst), Oruvail
(Wyeth-Ayerst), Ponstel (First Horizon), Relafen (GlaxoSmithI~line), Tolectin
(Ortho-McNeil), Toradol (Roche Labs., Inc.), Vioxx (Merck), Voltaren
(Novartis), Advair (GlaxoSmithI~line), Flovent (GlaxoSmithI~line), Pulmicort
(AstranZeneca), and Vanceril (Schering), Asacol (Procter & Gamble), Colazal
(Salix), Dipentum (Pharmacia & Upjohn), and Rowasa (Solvay).
In yet another embodiment, the compounds of the present invention may
be admistered in combination with an antirheumatic agent. Commercially
available antirheumatic agents include, but are not limited to, Anaprox (Roche
Labs.), Arava (Aventic), Arthrotec (Searle), Azulfidine (Pharmacia & Upjohn),
I S Cataflam (Novartis), Celebrex (Pfizer), Celestone (Schering), Cuprimine
(Merck), Enbrel ~(Immunex), Feldene (Pfizer), Gengraf (Abbott), Indocin
(Merck), Lodine (Wyeth-Ayerst), Naprosyn (Ruche Labs.), Neural (Novartis),
Pediapred (Celltech), Prednisone (Roxanne), Remicade (Centocor), Solu-Medrol
(Pharmacia & Upjohn), Triliate (Purdue Frederick), and Voltaren (Novartis).
Moreover, the compounds of the present invention may be used in
combination with any cardiovascular agent including, but not limited to,
adrenergic blockers such as Cardura (Pfizer), Dibenzyline (WellSpring), Hytrin
(Abbott), Minipress (Pfizer), and Minizide (Pfizer); adrenergic stimulants
such as
Aldoclor (Merck), Aldomet (Merck), Aldoril (Merck), Catapres (Boehringer
Ingelheim), Clorpres (Bertelc), and Tenex (Robins); alpha/beta adrenergic
blockers such as Coreg (GIaxoSmithl~line), and Normodyne (Schering);
angiotensin converting enzyme inhibitors such as Accupril (Parke-Davis), Aceon
(Solvay), Altace (Monarch), Captopril (Mylan), Enalaprilat (Baxter
Anesthesia),
Lotensin (Novartis), Mavik (Abbott), Monopril (Bristol-Myers Squibb), Prinivil
(Merck), Univasc (Schwarz), Vaotec (Merck), and Zestril (AstraZeneca);
angiotenisin converting enzyme inhibitors such as Lexxel (AstraZeneca), Lotrel
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(Novartis), Tarlca (Abbott), Accuretic (Parke-Davis), Lotensin (Novartis),
Prinzide (Merck), Uniretic (Schwarz), Vaeretic (Merck), and Zestoretic
(AstraZeneca); angiotensin II receptor antagonists such as Atacand
(AstraZeneca), Avapro (Briston-Myers Squibb), Cozaar (Merck), Diovan
(Novartis), Micardis (Boehringer Ingelheim), and Teveten (Unimed);
antiarrhythmics (Groups I-IV), antilipemic agents such as bile acid
sequestrants,
fibric acid derivatives, HMG-CoA reductase inhibitors, and nicotinic acid;
Beta
adrenergic blocking agents; calcium channel blockers; inotropic agents;
vasodilators including coronoary vasodilators, natriuretic peptides, and
peripheral
vasodilators; and vasopressors.
In another aspect of the present invention, the therapeutic agent comprises
a small molecule toxin, including maytansine, calicheamicin, trichothene, and
CC
1065. In a specific embodiment, the therapeutic agent may comprise one~more
calicheamicin molecules. The calicheamicin family of antibiotics are capable
of
producing double-stranded DNA breaks at sub-picomolar concentrations.
Structured analogues of calicheamicin are also known. See Hinman et al., 53
CANCER RESEARCH 3336-42 (1993); Lode et al., 58 CANCER RESEARCH 2925-28
(l 998).
In yet another aspect of the present invention, the therapeutic agent may
comprise one or more enzymatically active toxins and fragments thereof.
Examples of such toxins include nonbinding active fragments of diphtheria
toxin,
diphtheria A chain, exotoxin A chain (from Pseudomonas aeruginosa), ricin A
chain, abrin A chain, modeccin A chain, alpha-sarcin, dianthin proteins,
Phytolaca americana proteins (PAPI, PAPAII, and PAP-S), momordica charantia
inhibitor, curcin, crotin sapaonaria officinalis inhibitor, gelonin,
mitogellin,
restrictoein, phenomvcin, enomycin and the tricothecenes. See, e.g., WO
93/21232.
The present invention further contemplates therapeutic agents that have
nucleolytic activity such as a ribonuclease and a deoxyribonuclease. In
addition,
a variety of radioactive isotopes are available for the production of
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radioconjugated binding partners. Examples include Y9°, Atzzz, Ret86,
Re~86,
Sm~53, giz~z, P3z and radioactive isotopes of Lu.
1n yet another aspect of the present invention, the at least one compound
may be conjugated to a receptor, such as streptavidin, for utilization in
tumor
pretargeting. Briefly, the compound-receptor conjugate is administered to the
patient and unbound conjugate is removed from circulation with a clearing
agent.
A ligand, such as biotin, which is conjugated to a cytotoxic agent is then
administered.
Timing of Administration
1n several embodiments of the present invention, a compound described
herein is administered before or after administration of a second therapeutic
agent. The administration of a compound may occur anytime from several
minutes to several hours before the administration of the second therapeutic
agent. The compound may alternatively be administered anytime from several
hours to several days, possibly several weeks, and up to several months before
the second therapeutic agent.
More specifically, a compound of the present invention may be
administered at least about 1 minute, at least about minutes, at least about
minutes, at least about minutes, at least about minutes, at least about 2
minutes,
at least about 3 minutes, at least about 4 minutes, at least about 5 minutes,
at least
about 6 minutes, at least about 7 minutes, at least about 8 minutes, at least
about 9
minutes, at least about 10 minutes, at least about 11 minutes, at least about
12
minutes, at least about 13 minutes, at least about 14 minutes, at least about
15
minutes, at least about 16 minutes, at least about 17 minutes, at least about
18
minutes, at least about 19 minutes, at least about 20 minutes, at least about
21
minutes, at least about 22 minutes, at least about 23 minutes, at least about
24
minutes, at least about 25 minutes, at least about 26 minutes, at least about
27
minutes, at least about 28 minutes, at least about 29 minutes, at least about
30
minutes, at least about 31 minutes, at least about 32 minutes, at least about
33
minutes, at least about 34 minutes, at least about 35 minutes, at least about
36
minutes, at least about 37 minutes, at least about 38 minutes, at least about
39
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minutes, at least about at least about 41 minutes,
40 minutes, at least about 42
minutes, at least about at least about 44 minutes,
43 minutes, at least about 45
minutes, at least about at least about 47 minutes,
46 minutes, at least about 48
minutes, at least about at least about 50 minutes,
49 minutes, at least about 51
minutes, at least aboutat least about 53 minutes,
52 minutes, at least about 54
minutes, at least about at least about 56 minutes,
55 minutes, at least about 57
minutes, at least about
58 minutes, at least
about 59 minutes, or
at least about 60
minutes before or after
the second therapeutic
agent. Furthermore, a
compound of the present invention may be administered at least about 1 hour,
at
least about 2 hours, at least about 3 hours, at least about 4 hours, at least
about 5
hours, at least about 6 hours, at least about 7 hours, at least about 8 hours,
at least
about 9 hours, at least about 10 hours, at least about 11 hours, at least
about 12
hours, at least about 13 hours, at least about 14 hours, at least about 15
hours, at
least about 16 hours, at least about 17 hours, at least about 18 hours, at
least about
I S 19 hours, at least about 20 hours, at least about 21 hours, at least about
22 hours,
at least about 23 hours, or at least about 24 hours before or after the second
therapeutic agent.
Moreover, a compound of the present invention may be administered at
least about 1 day, at least about 2 days, at least about 3 days, at least
about 4
days, at least about 5 days, at least about 6 days, at least about 7 days, at
least
about 8 days, at least about 9 days, at least about 10 days, at least about 11
days,
at least about 12 days, at least about 13 days, at least about 14 days, at
least about
I S days, at least about 16 days, at least about 17 days, at least about 18
days, at
least about 19 days, at least about 20 days, at least about 21 days, at least
about
22 days, at least about 23 days, at least about 24 days, at least about 25
days, at
least about 26 days, at least about 27 days, at least about 28 days, at least
about
29 days, at least about 30 days or at least about 31 days before or after the
administration ofthe second therapeutic agent.
In yet another aspect of the present invention, a compound of the present
invention may be administered at least about 1 week, at least about 2 weeks,
at
least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least
about
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6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9
weeks, at
least about 10 weeks, at least about 1 I weeks, at least about 12 weeks, at
least
about 13 weeks, at least about 14 weeks, at least about 15 weeks, at least
about 16
weeks, at least about 17 weeks, at least about 18 weeks, at least about 19
weeks,
or at least about 20 weeks before or after the second therapeutic agent.
In a further aspect of the present invention, a compound of the present
invention may be administered at least about one month, at least about two
months, at least about three months, at least about four months, at least
about five
months, at least about six months, at least about seven months, at least about
eight months, at least about nine months, at least about ten months, at least
about
eleven months, or at least about twelve months before or after the second
therapeutic agent.
For convenience, the meaning of certain terms and phrases employed in
the specification, examples, and appended claims are provided below.
DEFINITIONS
As used herein, the term "compound" includes both the singular and the
plural, and includes any single entity or combined entities that have at least
the
activity disclosed herein and combinations, fragments, analogs or derivatives
of
such entities. Such entities include, but are not limited to, chemical
elements,
molecules, compounds, mixtures, emulsions, chemotherapeutic agents,
pharmacological agents, hormones, antibodies, growth factors, cellular
factors,
nucleic acids, proteins, peptides, peptidomimetics, nucleotides,
carbohydrates,
and combinations, fragments, analogs or derivatives of such entities.
The term "phenylamine" refers to a primary or secondary
benzeneamine, more commonly known as an aniline. The amino group on the
aniline can be substituted with hydrogen, alkyl (CI-CI~, straight chain or
branched), cycloalkyl (C3-Cio), or aryl substituted aryl groups. The phenyl
ring
of this aniline derivative can be optionally substituted with one or more
functional groups, or a combination of functional groups such as alkyl,
alkenyl,
alkynyl, phenyl, benzyl, halo, cyano, nitro, hydroxy, thioxy, alkoxy, aryloxy,
haloalkyloxy, alleylthio, arylthio, amino, alkyl amino, aryl amino, acyl,
carboxyl,
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amido, sulfonamido, sulfonyl, sulfate, sulfonic acid, morpholino, piperazinyl,
pyridyl, thienyl, furanyl, pyrroyl, pyrazoyl, phosphate, phosphonic acid, or
phosphonate. If applicable, these groups can be represented in protected or
unprotected forms used in standard organic synthesis.
The term "naphthylamine" refers to a primary or secondary a- or
[3-naphthylamine. The ring substructure in the naphthylamine can be optionally
substituted with one or a combination of functional groups such as alkyl,
alkenyl,
alkynyl, phenyl, benzyl, halo, cyano, nitro, hydroxy, thioxy, alkoxy, aryloxy,
haloalkyloxy, allcylthio, arylthio, amino, alkyl amino, aryl amino, acyl,
carboxyl,
amido, sulfonamido, sulfonyl, sulfate, sulfonic acid, morpholino,
thiomorpholino,
piperazinyl, pyridyl, thienyl, furanyl, pyrroyl, pyrazoyl, phosphate,
phosphonic
acid, phosphonate and the like. These groups can be represented in protected
or
unprotected forms used in standard organic synthesis.
The term "naphthylalkyl amine" refers to a primary or secondary
a- and (3-naphthylalkyl amine (for example, 2-a-naphthylethyl amine). The term
"benzalkyl amine" refers to a primary or secondary benzylalkyl amine (for
example, phenylethyl amine). These aryl alkyl substructures or compounds can
be optically active or optically inactive. The aryl (ring) substructures of
the
naphthylalkyl and benzalkyl amines can be optionally subsituted with one or a
combination of functional groups, such as alkyl, alkenyl, alkynyl, phenyl,
benzyl,
halo, cyano, nitro, hydroxy, thioxy, alkoxy, aryloxy, haloalkyloxy, alkylthio,
arylthio, amino, alkyl amino, aryl amino, acyl, carbolyl, amido, sulfonamido,
sulfonyl, sulfate, sulfonic acid, morpholino, piperazinyl, pyridyl, thienyl,
furanyl,
pyrroyl, pyrazoyl, phosphate, phosphonic acid, phosphonate and the like. If
applicable these groups can be represented in protected or unprotected forms
used
in standard organic synthesis.
The term "quinolinyl amine" refers to primary or secondary
quinolyl amines. These amines can be in optically active or inactive forms.
The
aryl (ring) substructure of the quinolyl amine can be be optionally
substituted
with one a combination of functional groups such as alkyl, alkenyl, alkynyl,
phenyl, benzyl, halo, cyano, nitro, hydroxy, thioxy, alkoxy, aryloxy,
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haloalkyloxy, alkylthio, arylthio, amino, alkyl amino, aryl amino, acyl,
carboxyl,
amide, sulfonamide, sulfonyl, sulfate, sulfonic acid, morpholino,
thiomorpholino,
piperazinyl, pyridyl, thienyl, furanyl, pyrroyl, pyrazoyl, phosphate,
phosphonic
acid, phosphonate and the like. These groups can be represented in protected
or
unprotected forms used in standard organic synthesis.
The teen "heteroaryl amines" refers to pyrroles, pyrazoles, imidazoles,
and indoles. The aryl (ring) substructure of the heteroaryl amine can be
optionally substituted with one or a combination of functional groups such as
alkyl, alkenyl, alkynyl, phenyl, benzyl, halo, cyano, nitre, hydroxy, thioxy,
alkoxy, aryloxy, haloalkyloxy, alkylthio, arylthio, amino, alkyl amino, aryl
amino, acyl, carboxyl, amide, sulfonamide, sulfonyl, sulfate, sulfonic acid,
morpholino, thiomorpholino, piperazinyl, phosphate, phosphonic acid, or
phosphonate. These groups can be represented in protected or unprotected forms
used in standard organic synthesis.
The teen "glycated protein," as used herein, includes proteins linked to
glucose, either enzymatically or non-enzymatically, primarily by condensation
of
free epsilon-amino groups in the protein with glucose, forming Amadori
adducts.
Furthermore, glycated protein, as used herein, includes not only proteins
containing these initial glycation products, but also glycation products
resulting
from further reactions such as rearrangements, dehydration, and condensations
that form irreversible advanced glycation end products (AGE).
The term "polynucleotide" refers generally to polymeric forms of
nucleotides of any length, either ribonucleotides or deoxynucleotides. Thus,
this
term includes, but is not limited to, single-stranded, double-stranded, or
multi-
stranded DNA or RNA. Polynucleotides may further comprise genomic DNA,
cDNA, or DNA-RNA hybrids. Moreover, the polynucleotides of the present
invention may be synthetically produced.
Polynucleotides may comprise chemically modified, biochemically
modified, or derivatized nucleotides. For example, a polynucleotide may
comprise, in part, modified nucleotides such as methylated nucleotides or
nucleotide analogs. In other embodiments, polynucleotides may comprise sugars,
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caps, nucleotide branches, and linking groups such as fluororibose and
thioate. In
addition, the sequence of nucleotides may be interrupted by non-nucleotide
components. Furthermore, a polynucleotide may be modified after
polymerization to facilitate its attachment to other polynucleotides,
proteins,
metal ions, labeling components, or a solid support.
The backbone of the polynucleotide may comprise modified or substituted
sugar and/or phosphate groups. Alternatively, the backbone of the
polynucleotide may comprise a polymer of synthetic subunits such as
phosphoramidites and thus may be an oligodeoxynucleoside phosphoramidate or
a mixed phosphoramidate-phosphodiester oligomer. See Peyrottes et al., NULL.
ACIDS RBS. (1996) 24:1841-1848, and Chaturvedi et al., Nucl.. AeIDS RES.
(1996) 24:2318-2323.
The term "homology", as used herein, refers to a degree of
complementarity. There may be partial homology or complete homology (i.e.,
I S identity). A partially complementary sequence is one that at least
partially
inhibits an identical sequence from hybridizing to a target polynucleotide; it
is
referred to using the functional term "substantially homologous." The
inhibition
of hybridization of the completely complementary sequence to the target
sequence may be examined using a hybridization assay (Southern or Northern
blot, solution hybridization and the like) under conditions of low stringency.
A
substantially homologous sequence or probe will compete for and inhibit the
binding (i.e., the hybridization) of a completely homologous sequence or probe
to
the target sequence under conditions of low stringency. This is not to say
that
conditions of low stringency are such that non-specific binding is permitted;
low
stringency conditions require that the binding of two sequences to one another
be
a specific (i.e., selective) interaction. The absence of non-specific binding
may
be tested by the use of a second target sequence which lacks even a partial
degree
of complementarity (e.g., less than about 30% identity); in the absence of non
specific binding, the probe will not hybridize to the second non-complementary
target sequence.
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The term "gene" refers to a polynucleotide sequence that comprises
coding sequences necessary for the production of a polypeptide or precursor,
and
may also include expression control sequences or other control or regulatory
sequences. The polypeptide can be encoded by a full length coding sequence or
by any portion of the coding sequence. The gene may be derived in whole or in
part from any source lenown to those of ordinary skill in the art including a
plant,
a fungus, an animal, a bacterial genome or episome, eukaryotic, nuclear or
plasmid DNA, cDNA, viral DNA, or chemically synthesized DNA. A gene may
constitute an uninterrupted coding sequence or it may include one or more
introns, bound by the appropriate splice junctions. Moreover, a gene may
contain
one or more modifications in either the coding or the untranslated regions
that
could affect certain properties of the polynucleotide or polypeptide, such as
the
biological activity or the chemical structure of the expression product, the
rate of
expression, or the manner of expression control. Such modifications include,
but
are not limited to, mutations, insertions, deletions, and substitutions of one
or
more nucleotides. In this regard, such modified genes may be referred to as
variants of the native gene.
"Gene expression" refers to the process by which a polynucleotide
sequence undergoes successful transcription and translation such that
detectable
levels of the nucleotide sequence are expressed as proteins or the
polynucleotide
sequence undergoes transcription , if RNA is copied from DNA, or replication
if
DNA is copied from DNA, such that the resulting nucleotide copies are
detectable.
The term "gene expression profile" refers to a group of genes representing
a particular cell or tissue type (e.g., neuron, coronary artery endothelium,
or
disease tissue) in any activation state. In one aspect, a gene expression
profile is
generated from cells exposed to a compound of the present invention. This
profile may be compared to a gene expression profile generated from the same
type of cell or tissue type prior to treatment with a compound of the present
invention. Furthermore, a series of gene expression profiles may be generated
from cells or tissues treated with a compound of the present invention,
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specifically, at different doses or a time-course to assess the effects of the
compound. A gene expression profile is also known as a gene expression
signature.
The term "differential expression" refers to both quantitative as well as
qualitative differences in the temporal and tissue expression patterns of a
gene.
For example, a differentially expressed gene may have its expression activated
or
completely inactivated in normal versus disease conditions. Such a
qualitatively
regulated gene may exhibit an expression pattern within a given tissue or cell
type that is detectable in either control or disease conditions, but is not
detectable
in both. "Differentially expressed polynucleotide," as used herein, refers to
a
polynucleotide sequence that uniquely identifies a differentially expressed
gene
so that detection of the differentially expressed polynucleotide in a sample
is
correlated with the presence of a differentially expressed gene in a sample.
Similarly, a differentially expressed protein may have its expression
activated or completely inactivated in normal versus disease conditions. Such
a
qualitatively regulated protein may exhibit an expression pattern within a
given
tissue or cell type that is detectable in either control or disease
conditions, but is
not detectable in both. A "differentially expressed protein," as used herein,
refers
to an amino acid sequence that uniquely identifies a differentially expressed
protein so that detection of the differentially expressed protein in a sample
is
correlated with the presence of a differentially expressed protein in a
sample.
"Cell type," as used herein, refers to a cell from a given source (e.g.,
tissue or organ), a cell in a given state of differentiation, or a cell
associated with
a given pathology or genetic makeup.
The term "polypeptide" refers to a polymeric form of amino acids of any
length, which may include translated, untranslated, chemically modified,
biochemically modified, and derivatized amino acids. A polypeptide may be
naturally occurring, recombinant, or synthetic, or any combination of these.
Moreover, the term "polypeptide," as used herein, refers to proteins,
polypeptides, and peptides of any size, structure, or function. For example, a
polypeptide may comprise a string of amino acids held together by peptide
bonds.
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A polypeptide may alternatively comprise a long chain of amino acids held
together by peptide bonds. Moreover, a polypeptide may also comprise a
fragment of a naturally occurring protein or peptide. A polypeptide may be a
single molecule or may be a multi-molecular complex. In addition, such
polypeptides may have modified peptide backbones as well.
The term "polypeptide" further comprises immunologically tagged
proteins and fusion proteins, including, but not limited to, fusion proteins
with a
heterologous amino acid sequence, fusion proteins with heterologous and
homologous leader sequences, and fusion proteins with or without N-terminal
methionine residues.
The term "protein expression" refers to the process by which a
polynucleotide sequence undergoes successful transcription and translation
such
that detectable levels of the amino acid sequence or protein are expressed.
The term "protein expression profile" refers to a group of proteins
representing a particular cell or tissue type (e.g., neuron, coronary artery
endothelium, or disease tissue). In one aspect, a protein expression profile
is
generated from cells or tissues exposed to a compound of the present
invention.
This profile may be compared to a protein expression profile generated from
the
same type of cell or tissue prior to treatment with a compound of the present
invention. Furthermore, a series of protein expression profiles may be
generated
from cells or tissues treated with a compound of the present invention,
specifically, at different doses or a time-course to assess the effects of the
compound. A protein expression profile is also known as a "protein expression
signature.'.'
As used herein, a "biomolecule" includes polynucleotides and
polypeptides. Moreover, a "biomolecular sequence," as used herein, is a term
that refers to all or a portion of a polynucleotide sequence. A biomolecular
sequence may also refer to all or a portion of a polypeptide sequence. Tn the
context of biomolecule, for example, perlecan, the term "functional
equivalent"
refers to a protein or polynucleotide molecule that possesses functional or
structural characteristics that are substantially similar to all or part of
the native
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perlecan protein or native perlecan-encoding polynucleotides. A functional
equivalent of a native perlecan protein may contain modifications depending on
the necessity of such modifications for a specific structure or the
performance of
a specific function. The teen "functional equivalent" is intended to include
the
"fragments," "mutants," "derivatives," "alleles," "hybrids," "variants,"
"analogs,"
or "chemical derivatives" of native perlecan.
A "host cell," as used herein, refers to a microorganism, a prokaryotic
cell, a eukaryotic cell or cell line cultured as a unicellular entity that may
be, or
has been, used as a recipient for a recombinant vector or other transfer of
polynucleotides, and includes the progeny of the original cell that has been
transfected. It is understood that the progeny of a single cell may not
necessarily
be completely identical in morphology or in genomic or total DNA complement
as the original parent due to natural, accidental, or deliberate mutation.
In the context of immunoglobulins, the term "functional equivalent"
refers to immunoglobulin molecules that exhibit immunological binding
properties that are substantially similar to the parent immunoglobulin. As
used
herein, the term "immunological binding properties" refers to non-covalent
interactions of the type which occur between an immunoglobulin molecule and
an antigen for which the immunoglobulin is specific. Indeed, a functional
equivalent of a monoclonal antibody immunoglobulin, for example, may inhibit
the binding of the parent monoclonal antibody to its antigen. A functional
equivalent may comprise F(ab')2 fragments, Flab) molecules, Fv fragments,
single chain fragment variable displayed on phage (scFv), single domain
antibodies, chimeric antibodies, or the like so long as the immunoglobulin
exhibits the characteristics of the parent immunoglobulin.
As used herein, the term "isolated" refers to a polynucleotide, a
polypeptide, an antibody, or a host cell that is in an environment different
from
that in which the polynucleotide, the polypeptide, the antibody, or the host
cell
naturally occurs. An isolated polynucleotide, polypeptide, antibody, or host
cell
is generally substantially purified.
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As used herein, the term "substantially purified" refers to a compound
that is removed from its natural environment and is at least about 60% to
99.9%
free from other components, or is at least about 60% free, at least about 65%
free,
at least about 70% free, at least about 75% free, at least about 80% free, at
least
about 83% free, at least about 85% free, at least about 88% free, at least
about
90% free, at least about 91 % free, at least about 92% free, at least about
93%
free, at least about 94% free, at least about 95% free, at least about 96%
free, at
least about 97% free, at least about 98% free, at least about 99% free, at
least
about 99.9% free, or at least about 99.99% free from other components with
which it is naturally associated. For example, a composition containing A is
"substantially free of B when at least about 85% by weight of the total A+B in
the composition is A. Alternatively, A comprises at least about 90% by weight
of
the total of A+B in the composition, further still, at least about 95% or even
99%
by weight.
"Diagnosis," as used herein, generally includes a determination of a
subject's susceptibility to a disease or disorder, a determination as to
whether a
subject is presently affected by a disease or disorder, a prognosis of a
subject
affected by a disease or disorder (e.g., identification of pre-metastatic or
metastatic cancerous states, stages of cancer, or responsiveness of cancer to
therapy), and therametrics (e.g., monitoring a subject's condition to provide
information as to the effect or efficacy of therapy).
The term "biological sample" encompasses a variety of sample types
obtained from or originating from an organism which may be used in diagnostic,
monitoring, or other assays. The term encompasses blood, serum, plasma, cells,
proteins, carbohydrates, nucleic acids, urine, nasal secretions, mucosal
secretions,
cellular fluid, cellular exudate and other liquid samples of biological
origin, solid
tissue samples such as a biopsy specimen, or tissue cultures or cells derived
therefrom and the progeny thereof. The term specifically encompasses a
clinical
sample, and further includes cells in cell culture, cell supernatants, cell
lysates,
amniotic fluid, biological fluids, and tissue samples. The term also
encompasses
samples that have been manipulated in any way after procurement such as
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treatment with reagents, solubilization, or enrichment for certain components.
The biological sample can be derived from the organism directly or can be
collected from the environment.
The terms "individual," "subject," "host," and "patient" refer to any
subject for whom diagnosis, treatment, or therapy is desired. In one
embodiment,
the individual, subject, host, or patient is a human. Other subjects may
include,
but are not limited to, animals including but not limited to, cattle, sheep,
horses,
dogs, cats, guinea pigs, rabbits, rats, primates, opossums and mice. Other
subjects include species of bacteria, phages, cell cultures, viruses, plants
and
other eucaryotes, prokaryotes and unclassified organisms.
The terms "treatment," "treating," "treat," and the like are used herein to
refer generally to obtaining a desired pharmacological and/or physiological
effect. The effect may be prophylactic in terms of completely or partially
preventing a disease or symptom thereof and/or may be therapeutic in terms of
a
IS partial or complete stabilization or cure for a disease and/or adverse
effect
attributable to the disease. "Treatment" as used herein covers any treatment
of a
disease in a subject, particularly a human, and includes: (a) preventing the
disease
or symptom from occurring in a subject which may be predisposed to the disease
or symptom, but has not yet been diagnosed as having it; (b) inhibiting the
disease symptom, i.e., arresting its development; or (c) relieving the disease
symptom, i.e., causing regression of the disease or symptom.
The expression "therapeutically effective amount" refers to an amount of,
for example, a compound disclosed herein, that is effective for preventing,
ameliorating, treating or delaying the onset of a disease or condition.
A "prophylactically effective amount" refers to an amount of, for
example, a compound disclosed herein that is effective for preventing a
disease or
condition.
A "liposome" is a small vesicle composed of various types of lipids,
phospholipids andlor surfactant, which is useful for delivery of a drug to a
subject, such as a mammal or other animal. The compounds of the present
invention may be delivered by a liposome. The components of the liposome are
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commonly arranged in a bilayer formation, similar to the lipid arrangement of
biological membranes. Liposome formulations, loading of liposomes and
administration and delivery of liposomes are known in the art.
"Hybridization," broadly defined, refers to any process by which a
polynucleotide sequence binds to a complementary sequence through base
pairing. Hybridization conditions can be defined by, for example, the
concentrations of salt or formamide in the prehybridization and hybridization
solutions, or by the hybridization temperature, and are well known in the art.
Hybridization can occur under conditions of various stringency. Hybridization
may also refer to the binding of a protein-capture agent to a target protein
under
certain conditions, such as normal physiological conditions.
As understood herein, the term "activation" refers to any alteration of a
signaling pathway or biological response including, for example, increases
above
basal levels, restoration to basal levels from an inhibited state, and
stimulation of
the pathway above basal levels.
The term "biological activity" refers to the biological behavior and effects
of a protein or peptide. The biological activity of a protein may be affected
at the
cellular level and the molecular level. For example, an antisense
oligonucleotide
may prevent translation of a particular mRNA, thereby inhibiting the
biological
activity of the protein encoded by the mRNA. In addition, an antibody may bind
to a particular protein and inhibit that protein's biological activity.
The term "oligonucleotide" as used herein refers to a polynucleotide
sequence comprising, for example, from about 4 nucleotides (nt) to about 1000
nt. Oligonucleotides for use in the present invention are preferably from
about 15
nt to about 150 nt, more preferably from about I50 nt to about 1000 nt in
length.
The oligonucleotide may be a naturally occurring oligonucIeotide or a
synthetic
oligonucleotide. Oligonucleotides may be prepared by the phosphoramidite
method (Beaucage and Carruthers, TETRAHEDRON LETT. (1981) 22:1859-1862),
or by the triester method (Matteucci et al., J. AM. CHEM. Soe. (1981)
103:3185),
or by other chemical methods known in the art.
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The term "microarray" refers generally to the type of genes or proteins
represented on a microarray by oligonucleotides (polynucleotide sequences) or
protein-binding agents, and where the type of genes or proteins represented on
the microarray is dependent on the intended purpose of the microarray (e.g.,
to
monitor expression of human genes or proteins). The oligonucleotides or
protein-binding agents on a given microarray may correspond to the same type,
category, or group of genes or proteins. Genes or proteins may be considered
to
be of the same type if they share some common characteristics such as species
of
origin (e.g., human, mouse, rat); disease state (e.g., cancer); function
(e.g.,
protein kinases, tumor suppressors); same biological process (e.g., apoptosis,
signal transduction, cell cycle regulation, proliferation, differentiation).
For
example, one microarray type may be a "cancer microarray" in which each of the
microarray oligonucleotides or protein-binding agents correspond to a gene or
protein associated with a cancer. An "epithelial microarray" may be a
microarray
of oligonucleotides or protein-binding agents corresponding to unique
epithelial
genes or proteins. Similarly, a "cell cycle microarray" may be an microarray
type in which the oligonucleotides or protein-binding agents correspond to
unique genes or proteins associated with the cell cycle.
The term "detectable", one in sense, refers to a polynucleotide expression
pattern which is detectable via the standard techniques of polymerase chain
reaction (PCR), reverse transcriptase (RT) -PCR (RT-PCR), differential
display,
and Northern analyses, which are well known to those of skill in the art.
Similarly, polypeptide expression patterns may be "detected" via standard
techniques including immunoassays such as Western blots. In .general, the term
"detectable is used when a result of an action, such as addition of a compound
in
an assay step, is observable, particularly by physical means, such as a color
change.
A "target gene" refers to a polynucleotide, often derived from a biological
sample, to which an oligonucleotide probe is designed to specifically
hybridize.
It is either the presence or absence of the target polynucleotide that is to
be
detected, or the amount of the target polynucleotide that is to be quantified.
The
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target polynucleotide has a sequence that is complementary to the
polynucleotide
sequence of the corresponding probe directed to the target. The target
polynucleotide may also refer to the specific subsequence of a larger
polynucleotide to which the probe is directed or to the overall sequence
(e.g.,
gene or mRNA) whose expression level it is desired to detect.
A "target protein" refers to an polypeptide, often derived from a
biological sample, to which a protein-capture agent specifically hybridizes or
binds. It is either the presence or absence of the target protein that is to
be
detected, or the amount of the target protein that is to be quantified. The
target
protein has a structure that is recognized by the corresponding protein-
capture
agent directed to the target. The target protein or amino acid may also refer
to the
specific substructure of a larger protein to which the protein-capture agent
is
directed or to the overall structure (e.g., gene or mRNA) whose expression
level
it is desired to detect.
IS The term "complementary" refers to the topological compatibility or
matching together of the interacting surfaces of a probe molecule and its
target.
The target and its probe can be described as complementary, and furthermore,
the
contact surface characteristics are complementary to each other. Hybridization
or
base pairing between nucleotides or nucleic acids, such as, for example,
between
the two strands of a double-stranded DNA molecule or between an
oligonucleotide probe and a target are complementary.
The term "background" refers to non-specific binding or other
interactions between, for example, polynucleotides, polypeptides, small
molecules and polypeptides, or small molecules and polynucleotides.
"Background" may also refer to the non-specific binding or other interactions
in
the context of assays including immunoassays.
In the context of microarrays, the term "background" refers to
hybridization signals resulting from non-specific binding, or other
interactions,
between the labeled target polynucleotides and components of the
oligonucleotide microarray (e.g., the oligonucleotide probes, control probes,
the
microarray support) or between target proteins and the protein-binding agents
of
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a protein microarray. Background signals may also be produced by intrinsic
fluorescence of the microarray components themselves. A single background
signal may be calculated for the entire microarray, or a different background
signal may be calculated for each target polynucleotide or target protein. The
background may be calculated as the average hybridization signal intensity, or
where a different background signal is calculated for each target gene or
target
protein. Alternatively, background may be calculated as the average
hybridization signal intensity produced by hybridization to probes that are
not
complementary to any sequence found in the sample (e.g., probes directed to
polynucleotides of the opposite sense or to genes not found in the sample such
as
bacterial genes where the sample is mammalian polynucleotides). The
bacleground can also be calculated as the average signal intensity produced by
regions of the microarray which lack any probes or protein-binding agents at
all.
A "small molecule" comprises a compound or molecular complex, either
synthetic, naturally derived, or partially synthetic, composed of carbon,
hydrogen, oxygen, and nitrogen, which may also contain other elements, and
which may have a molecular weight of less than about 100 to about 15,000
Daltons, or less than about 15,000, less than about 14,000, less than about
13,000,
less than about 12,000, less than about I 1,000, less than about 10,000, less
than
about 9,000, less than about 8,000, less than about 7,000, less than about
6,000,
less than about 5,000, less than about 4,000, less than about 3,000, less than
about
2,000, less than about 1,000, less than about 900, less than about 800, less
than
about 700, less than about 600, less than about 500, less than about 400, less
than
about 300, less than about 200, or less than about 100.
The term "fusion protein" refers to a protein composed of two or more
polypeptides that, although typically not joined in their native state, are
joined by
their respective amino and carboxyl termini through a peptide linkage to form
a
single continuous polypeptide. It is understood that the two or more
polypeptide
components can either be directly joined or indirectly joined through a
peptide
linker/spacer.
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The term "normal physiological conditions" means conditions that are
typical inside a living organism or a cell. Although some organs or organisms
provide extreme conditions, the infra-organismal and infra-cellular
environment
normally varies around pH 7 (i.e., from pH 6.5 to pH 7.5), contains water as
the
predominant solvent, and exists at a temperature above 0°C and below
50°C. The
concentration of various salts depends on the organ, organism, cell, or
cellular
compartment used as a reference.
The term "cluster" refers to a group of clones or biomolecular sequences
related to one another by sequence homology. In one example, clusters are
formed based upon a specified degree of homology and/or overlap (e.g.,
stringency). "Clustering" may be performed with the sequence data. For
instance, a biomolecular sequence thought to be associated with a particular
molecular or biological activity in one tissue might be compared against
another
library or database of sequences. This type of search is useiiil to look for
homologous, and presumably functionally related, sequences in other tissues or
samples, and may be used to streamline the methods of the present invention in
that clustering may be used within one or more of the databases to cluster
biomolecular sequences prior to performing a method of the invention. The
sequences showing sufficient homology with the representative sequence are
considered part of a "cluster." Such "sufficient" homology may vary within the
needs of one skilled in the art.
As used herein, the term "internal database" refers to a database
maintained within a local computer network. It contains, for example,
biomolecular sequences associated with a project. It may also contain
information associated with sequences including, but not limited to, a library
in
which a given sequence is found and descriptive information about a likely
gene
associated with the sequence. The internal database may typically be
maintained
as a private database behind a firewall within an enterprise network. However,
the invention is not limited to only this embodiment and an internal database
could be made available to the public. The internal database may include
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sequence data generated by the same enterprise that maintains the database,
and
may also include sequence data obtained from external sources.
The term "external database," as understood herein, refers to a database
located outside all internal databases. Typically, an enterprise network
differing
from the enterprise network maintaining the internal database will maintain an
external database. The external database may be used, for example, to provide
some descriptive information on biomolecular sequences stored in the internal
database. In one embodiment, the external database is GenBank and associated
databases maintained by the National Center for Biotechnology Information
(NCBI), part of the National Library of Medicine.
As used herein and in the appended claims, the singular forms "a," "an,"
and "the" include plural reference unless the context clearly indicates
otherwise.
Thus, for example, reference to a "compound" is a reference to one or more
such
compounds and includes equivalents thereof known to those skilled in the art,
and
so forth.
Unless defined otherwise, all technical and scientific terms used herein
have the same meaning as commonly understood to one of ordinary skill in the
art to which this invention belongs. Although any methods, devices, and
materials similar or equivalent to those described herein can be used in the
practice or testing of the invention, the preferred methods, devices and
materials
are now described.
All publications and patents mentioned herein are incorporated herein by
reference for the purpose of describing and disclosing, for example, the
constructs and methodologies that are described in the publications, which
might
be used in connection with the presently described invention. 'The
publications
discussed above and throughout the text are provided solely for their
disclosure
prior to the filing date of the present application. Nothing herein is to be
construed as an admission that the inventors are not entitled to antedate such
disclosure by virtue of prior invention.
It is to be understood that this invention is not limited to the particular
methodology, protocols, cell lines, constructs, and reagents described herein
and
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as such may vary. It is also to be understood that the terminology used herein
is
for the purpose of describing particular embodiments only, and is not intended
to
limit the scope of the present invention which will be limited only by the
appended claims.
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EXAMPLES
The present invention is further illustrated by the following
examples, which are not to be construed in any way as imposing limitations
upon
the scope thereof, but rather are illustrative only. On the contrary, it is to
be
clearly understood that resort may be had to various other embodiments,
modifications, and equivalents thereof which, after reading the description
herein,
may suggest themselves to one of ordinary skill in the art without departing
from
the spirit of the present invention or the scope of the appended claims.
The following acronyms, abbreviations, terms and definitions have
been used throughout the experimental section. Acronyms or abbreviations:
DIEA (N,N diisopropylethylamine), THF (tetrahydrofuran), HPLC (high
performance liquid chromatography), TLC (thin layer chromatography), mp
(melting point), rt (room temperature), aq (aqueous), min (minute), h (hr,
hour),
atm (atmosphere), conc. (concentrated), MS (mass spectroscopy/spectrometry),
NMR (riuclear magnetic resonance), Rf (TLC retention factor), and Rt (HPLC
retention time). NMR abbreviations: br (broad), apt (apparent), s (singlet), d
(doublet), t (triplet), q (quartet), dq (doublet of quartets), dd (doublet of
doublets),
dt (doublet of triplets), m (multiplet).
EXAMPLE 1
General Synthetic, Purifieatio~, CharacteYization, and Spectroscopic
ProceduYes
General Synthetic Procedures. Room temperature is defined as an ambient
temperature range, typically 20-25 °C. An ice bath (crushed icelwater)
temperature is defined as a range, typically -5 to 0 °C. Temperature at
reflux is
defined as ~15 °C of the boiling point of the primary reaction solvent.
Overnight
is defined as a time range of 8-16 hours. Vacuum filtration (water aspirator)
is
defined as range of 5-I S mm Hg. Dried under vacuum is defined as using a high
vacuum pump as a range of 0.1-5 mm Hg. Neutralization is defined as a typical
acid-based neutralization method and measured to a pH 6-8 range using pH-
indicating paper. Brine is defined as a saturated aqueous sodium chloride.
Nitrogen atmosphere is defined as positive static pressure of nitrogen gas
passed
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through a Drierite column with an oil bubbler system. Concentrated ammonium
hydroxide is defined as an approximately 15 M solution.
All eluents for column or thin layer chromatography were
prepared and reported as volume:volume (v:v) solutions, and HPLC eluent ratios
are v:v ratios. Aqueous sodium hydroxide or sodium bicarbonate solutions were
prepared as weight:volume (w:v) ratios. Aqueous hydrochloric acid solutions
were prepared as v:v ratios.
The quantities of solvents and/or reagents used for reaction work-
up or product isolation are those typically used by one trained in the art of
organic chemical synthesis, and the quantity of these solvents and/or reagents
used is determined based upon synthetic experience and appropriateness to the
specific reaction. For example: 1) crushed ice quantity ranged from about 10-
1000 g depending on reaction scale, 2) silica gel quantity used in column
chromatography depended on material quantity, complexity of mixture, and size
of chromatography column employed and ranged from about 5-1000 g, 3)
extraction solvent volume ranged from about 10-500 mL depending on reaction
size, 4) washes employed in compound isolation ranged from about 10-100 mL
of solvent or aq reagent depending on scale of reaction 5) drying reagents
(potassium carbonate, sodium carbonate or magnesium sulfate) ranged from
about 5-100 g depending on the amount of solvent to be dried and its water
content.
Melting points were measured against a mercury thermometer and
are not corrected.
For column chromatography employing concentrated ammonium
hydroxide as part of the mobile phase, the fractions collected from the column
were dried over sodium sulfate, potassium carbonate or a mixture of both. Then
the organic layer was filtered by gravity or vacuum to remove the drying agent
prior to concentration/evaporation.
Flash Chromatography. In the Tables, "ISCO" indicates
purification by flash chromatography as follows. Instrument: ISCO
CombiFlasha Si l Ox. Column: ISCO RediSepa - Disposable Columns for Flash
Chromatography (10 g of silica gel - normal phase - 35-60 micron particle size
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(230-400 mesh)). Mobile Phase A: CHZC12; Mobile Phase B: 10% NH40H in
MeOH; Gradient: 0-10% B in 22 min, hold 10% B for 18 min; Fractions: 30
fractions collected per column, 1.5 min each. Flow rate: 8.93 mL/min. The
salient fractions were analyzed by MS and TLC (90:9:1 CH2CIz:MeOH:NH40H
- Rt~ range 0.15 - 0.45) and combined in barcoded, tared vials. The resulting
solutions were sampled for LC/MS analysis, concentrated in vacuo and their
masses and yields were determined as tabulated in the Tables.
If no additional purification was carried out after completion of
the Parallel Synthesis, this is indicated as "None" in Table 2.
Analytical HPLC Procedures. Analytical HPLC procedures
were carried out according in one of two specific methods, depending upon
availability of instrumentation and sample requirements, as follows.
HPLC Method A. Column: Thomson Inst. Co. 4.6 x 50 mm CI8
5 l.un 60 A; Mobile Phase A: H20 with 0.1% TFA; Mobile Phase B: CH3CN
IS with 0.1% TFA; Detection: UV 254 nm. Gradient 1: ELSD12MG; 10-90% B
in.10 min, hold 90% B for 5 min; Flow: 1.0 mL/min. Gradient 2: ELSDSMG;
I S-100% B in 5 min, hold 100% B for 3 min; Flow -2.0 mL/min.
HPLC Method B. Colunzn: Thomson Inst. Co. 21 x 50 mm C18
5 l.un 60 A; Mobile Phase A: H20 with 0.1% TFA; Mobile Phase B: CHsCN
with 0.1 % TFA; Detection: UV 254 nm. Gradient 1: MIC8MG; 0-100% B in 8
min, hold 100% B for 2 min; Flow: 0.5 mL/min. Gradient 2: MIC15MG; 10-
90% B in 15 min, hold 90% B for 3 min; Flow: 0.5 mL/min.
Preparative HPLC Procedures. Preparative HPLC was carried
as follows. Instrument: Gilson; Column: Thomson Inst. Co. 21.5 x 150 mm
C18 5 pm 60 A; Mobile Phase A: H20; Mobile Phase B: CH3CN; Gradient:
I S-100% B in 10 min, hold 100% B for 5 min; Flow rate: 22 mL/min;
Detection: UV 254 nm. The fractions containing the desired compounds were
collected in barcoded, tared vials, sampled for LC/MS analysis, concentrated
ira
vaczro and their masses and yields were determined as shown in the Tables.
Spectroscopic and other Instrumental Procedures.
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NMR. The ~H and '3C NMR spectra described herein were obtained using
Varian INOVA600 (600MHz), Varian UNITY600 (600 MHz), or Varian 400
(400 MHz) spectrometers. Spectrometer field strength and NMR solvent used for
a particular sample are indicated in the Examples, or on any NMR spectra
actually shown as Figures. Typically, ~H NMR chemical shifts are reported as 8
values in parts per million (ppm) downfield from tetramethylsilane (TMS) (8 =
0
ppm) as an internal standard, and '3C NMR chemical shifts are reported in ppm
downfield from TMS and referenced with respect to the CDC13 signal center line
(8 = 77.0 ppm). Solid or liquid samples were dissolved in an appropriate NMR
solvent (CDCl3 or DMSO-d6), placed in a NMR sample tube, and data were
collected according to the spectrometer instructional manuals. Most samples
were analyzed in Variable Temperature mode, typically at about 55°C,
though
some data for some samples were collected with the probe at ambient
temperature. NMR data were processed using NUTS: NMR Utility Transform
Software (Lite Version-2001 I 128) by Acorn NMR.
LC-MS. The Liquid Chromatography-Mass Spectrometry (LC-
MS) instrumentation used to examine the compounds of the present invention
was typically a quadrupole/time-of flight mass spectrometer, with electrospray
ionization (ESI). For example, the typical LC-MS instrumentation used was a
Micromass Q-Tof using electrospray ionization (ESI). This instrument is a
quadrupole/time-of flight mass spectrometer capable of mass resolution up to
m/z
of about 7500. Samples were introduced in a direct injection mode by first
dissolving and diluting the sample in methanol or acetonitrile and injecting
the
sample solution into the ESI source via a IOpL loop Rheodyne injection valve.
The carrier solvent was typically a mixture of 70% CH3CN or MeOH and 30%
H20 (v:v), containing about 0.1% formic acid. Accurate mass analyses were
performed in a similar fashion except for using a multipoint mass calibration
with
the same instrument under high mass resolution conditions. Samples were spiked
with an appropriate internal mass reference compound, as known by one of
ordinary skill, and analyzed as described above.
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EXAMPLE 2
General Methods for Parallel Synthesis
Examples 3-5 describe the synthetic procedures for the preparation
of the "library" of Na, Nd, IV6-tris(amino)-1,3,5-triazines which was prepared
based on the strategy of changing only one pendant amino group per synthesis,
and based on the parent structure 95 shown below, where each compound in the
library contains two ofthe pendant groups in 95.
The library was divided into three subgroups, and all three
10 subgroups are presented in Table 2. Library I (compounds 1-50) includes
compounds having unchanged cycloheptylamino and [(1-ethyl-2-
pyrrolidinyl)methyl]amino substituents, with various groups being permuted at
the remaining triazine amino position, prepared by Method A as presented in
Example 3. Library II (compounds 51-75) includes compounds having
15 unchanged [(1-ethyl-2-pyrrolidinyl)methyl]amino and (3-fluoro-4-
methoxyphenyl)amino substituents, with various groups being permuted at the
remaining triazine amino position, prepared by Method B as presented in
Example 4. Library III (Compounds 76-100) includes compounds having
unchanged (3-fluoro-4-methoxyphenyl)amino arid cycloheptylamino substituents,
20 with various groups being permuted at the remaining triazine amino
position,
prepared by Method C as described in Example 5. Thus, the combination of the
specific amines employed produced a library of compounds of novel
composition. The sequence in which each monomer is added to form the
compounds of the library is also presented in Table 2, because the Monomer 1
25 amine is added first, Monomer 2 amine added second, and Monomer 3 amine is
added third.
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EXAMPLE 3
Par allel Synthetic Method A, for Library I Compounds
The following reaction scheme presents the general reagents and
conditions for parallel synthetic method A used for the compounds of Table 2
which designate Method A.
_Ar
CI
N~N a, b, c
~I
CI~N~CI
Reagents and conditions: (a) ArNHR, DIEA, CH3CN/1,4-dioxane, -11 C, 1h
(b) cycloheptylamine, DIEA, CH3CN/1,4-dioxane, rt, overnight
(c) 2-(aminomethyl)-1-ethylpyrrolidine, DIEA, CH3CN/1,4-dioxane, 80 C, 15
A stock solution of cyanuric chloride (0.542 M) in I,4-dioxane
was prepared and 1 mL of this solution (containing 100 mg or 0.542 mmol) was
dispensed into each of 50 barcoded 40 mL vials. These solutions were cooled to
about -1 I °C (freezing) using a J-KEM block connected to a circulating
cooler.
Meanwhile, individual solutions of each aryl amine ArNHR (specified as
Monomer I in Table 2, 0.542 mmol) and disopropylethylamine (DIEA) (77
mg/104 p.L, 0.596 mmol) in 1 mL of CH3CN were prepared. (For HCl salts, 204
p,L DIEA (approx. 2.1 equiv) was used.) Over a period of about 1 h, the
amine/DIEA solutions were added to the corresponding frozen cyanuric chloride
solutions, one by one, with swirling. The resulting solutions were then shaken
at
about -I I°C for about I h and the reaction block was allowed to warm
to room
temperature over the next hour. The resulting 2-amino-4,6-dichlorotriazine
solutions were carried to the next step without purification.
A stock solution of cycloheptylamine (1.08 M) and DIEA (1.19
M) in CH3CN was prepared and 0.5 mL (containing 6I mg l 69 pL, 0.542 mmol
amine and 77 mg ll04 yL, 0.596 mmol DIEA) was dispensed into each of the 40
mL vials from the first step. The vials were shaken on the J-KEM block
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overnight at room temperature and placed in a freezer (about -14 °C)
without
purification until the next reaction.
A stock solution of 2-(aminomethyl)-1-ethylpyrrolidine (1.08 M)
and DIEA (1.19 M) in CH3CN was prepared and 0.5 mL (containing 69 mg / 79
~L, 0.542 mmol amine and 77 mg /104 p.L, 0.596 mmol DIEA) was dispensed
into each of the 40 mL from the second step. The vials were then shaken on the
J-KEM block at about 80 °C for about I S h. The solutions were cooled
to room
temperature and taken to dryness ih vacuo. The residues were then extracted
with
ethyl acetate and the extract was washed with brine. The aqueous layers were
extracted a second time with ethyl acetate and the combined organic layers
were
dried over NaZS04 and passed through a plug of CeliteTM into barcoded, tared
vials. After concentration in vacuo, masses were determined and yields were
calculated, and the compounds were sampled for LC/MS analysis.
EXAMPLE 4
Parallel Synthetic Method B, for Library II C~mpounds
The following reaction scheme presents the general reagents and
conditions for parallel synthetic method B, used for the compounds of Table 2
which designate Method B.
CI
N~N a, b, c
~ RN
Cf~N~CI R2
~N
Reagents and conditions: (a) 3-fluoro-p-anisidine, DIEA, CH3CN11,4-dioxane,
-20 C, 1h (b) R2NHR, DIEA, CH3CN/1,4-dioxane, rt, overnight
(c) 2-(aminomethyl)-1-ethylpyrrolidine, DIEA, CH3CN/1,4-dioxane, 80 C, 15
In an oven-dried round bottom flask, a solution of cyanuric
chloride (5.0 g, 27.1 mmol) in 1,4-dioxane (40 mL) was cooled to freezing in a
CH3CN/dry ice bath. To this frozen solution was added 40 mL of CH3CN,
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followed by DIEA (3.85 g/ 5.19 mL, 29.8 mmol). A solution of 3-fluoro p
anisidine (3.83 g, 27.1 mmol) in 10 mL of CH3CN was then added slowly via
syringe. The reaction mixture was stirred at about -20 °C for about 1h
and
allowed to warm to room temperature over about 1h. The resulting 2-amino-4,6
dichlorotriazine solution was carried to the next step without purification.
Fifty mL (13.5 mmol) of the prepared (4,6-dichloro-[1,3,5]triazin-
2-yl)-(3-fluoro-4-methoxy-phenyl) amine solution was divided equally (2 mL or
0.54 mmol each) among 25 barcoded, 40 mL scintillation vials. Individual
solutions of each RaNHR (where R2 amine indicates Monomer 2 in Table 2,
0.542 mmol) and DIEA (77 mg /104 p.L, 0.596 mmol) in 0.5 mL of CH3CN were
prepared and added to the correspondingly labeled 40 mL vials. The resulting
solutions were shaken on the J-KEM block overnight at room temperature and
then placed in a freezer (about -14 °C) without purification until the
next
reaction.
A stocle solution of 2-(aminomethyl)-1-ethylpyrrolidine (1.08 M)
and DIEA (1.19 M) in CH3CN was prepared and 0.5 mL (containing 69 mg / 79
p,L, 0.542 mmol amine and 77 mg /104 p.L, 0.596 mmol DIEA) was dispensed
into each of the 40 mL vials from the second step. The vials were shaken on
the
J-KEM block at about 80 °C for about I S h. The solutions were cooled
to room
temperature and concentrated ire vacuo. The residues were then extracted with
ethyl acetate and the extract washed with brine. The aqueous layers were
extracted a second time with ethyl acetate and the combined organic layers
were
dried over Na2S04 and passed through a plug of CeliteTM into barcoded, tared
vials. After concentration i~ vacuo, masses were calculated and the compounds
were sampled for LC/MS analysis.
EXAMPLE 5
Parallel Sy~zthetic Method C, for Library III Compounds
The following reaction scheme presents the general reagents and
conditions for parallel synthetic method C, used for the compounds of Table 2
which designate Method C.
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F
OMe
HN
CI N~N
N~N a, b, c
~ HN N NR
CI~N~CI R3
Reagents and conditions: (a) 3-fluoro-p-anisidine, DIEA, CH3CNl1,4-dioxane,
-20 C, 1 h (b) cycloheptylamine, DIEA, CH3CN/1,4-dioxane, rt, overnight
(c) R3NHR, DIEA, CH3CN/1,4-dioxane, 00 C, 15
In an oven-dried round bottom flask, a solution of cyanuric
chloride (5.0 g, 27.1 mmol) in 1,4-dioxane (40 mL) was cooled to freezing in a
CH3CN/dry ice bath. To this frozen solution was added 40 mL of CH3CN,
followed by DIEA (3.85 g/ 5.19 mL, 29.8 mmol). A solution of 3-fluoro p-
anisidine (3.83 g, 27.1 mmol) in 10 mL of CH3CN was then added slowly via
syringe. The reaction mixture was stirred at about -20 °C for about 1h
and
allowed to warm to room temperature over 1h. The resulting 2-amino-4,6-
dichlorotriazine solution was carried to the next step without purification.
Fifty mL (13.5 mmol) of the prepared (4,6-dichloro-[1,3,5]triazin-
2-yl)-(3-fluoro-4-methoxy-phenyl) amine solution was treated with a solution
of
cycloheptylamine (1.53 g/1.73 mL, 13.5 mmol) and DIEA (1.93 g/2.60 mL, 14.9
mmol) in CH3CN (8 mL). The resulting solution was stirred overnight at room
temperature and carried to the next step without purification.
The resulting 6-chloro-N cycloheptyl-N'-(3-fluoro-4-methoxy-
phenyl)-[1,3,5]triazine-2,4-diamine solution (13.5 mmol) was diluted up to
62.5
mL with CH3CN and divided equally (2.5 mL or 0.54 mmol each) between 25
barcoded 40 mL scintillation vials. Individual solutions of each R3NHR (where
R3 amine indicates Monomer 3 in Table 2, 0.542 mmol) and DIEA (77 mg /104
p,L, 0.596 mmol) in 0.5 mL of CH3CN were prepared and added to the
correspondingly labeled 40 mL vial. The resulting solutions were shaken on the
J-KEM block at about 80 °C for about 15 h. The solutions were cooled
to room
temperature and concentrated in vacuo. The residues were then extracted with
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ethyl acetate and the extract washed with brine. Each organic layer was dried
over Na2S04 and passed through a plug of CeliteTM into a barcoded, tared vial.
After concentration in vacuo, masses were calculated and the compounds were
sampled for LC/MS analysis.
EXAMPLE 6
Synthesis of 6-Chlo~o-N (3-chloro-4-methoxy phenyl)-N'-cyclohexylmethyl-
~1,3,SJt~~iazine-2,4-dianai~e (102)
OCH3
OCH3 CI
CI I ~ I s
HN
N
HN NHS N ~~-CI
N
N N CI HN N
NaOH (aq), Hz0
G acetone, reflex, 102
101 3 hr, Na atm
H /~
tJ~N-CH3
GH ~--~3
NaOH (aq), H20
1,4-dioxane, acetone
reflex, 2 hr, NZ atm
I 0 103
To a sample of 101 (0.3004 g, 1.0 mmol, prepared as indicated
herein) dissolved in acetone (4 mL) was added a solution of
cyclohexanemethylamine (0.13 mL, 1.0 mmol) in acetone (1 mL) followed by
1 S addition of a NaOH solution (0.0448 g, 1.0 mmol dissolved in 1 mL of HaO).
The reaction mixture was allowed to stir at reflex for about 3 hours. The
reaction
mixture was then poured over crushed ice and neutralized with 10% HCl (aq) and
5% NaOH (aq). The resulting solid was collected by vacuum filtration, washed
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with water and dried overnight under vacuum to afford compound 102 (0.29 g,
76% recovery).
EXAMPLE 7
Synthesis of N (3-Chloro-4-rnethoxy phenyl)-N'-cyclohexylmethyl-N"-methyl-
N"-(1-rr~ethyl piperidin-4 yl)-~1,3,SJtriazine-2,4,6-triamine (103)
To a sample of 102 (0.286 g, 1.0 mmol) dissolved in 1,4-dioxane
(4 mL) was added a solution of N methyl-4(methylamino)piperidine (0.15 mL,
1.0 mmol) in acetone (I mL) followed by addition of a NaOH solution (0.0462 g,
1.0 mmol dissolved in 1 mL of HZO). The reaction mixture was allowed to stir
at
about 80°C for about 2 hours. The reaction mixture was poured over
crushed ice
and neutralized with 10% HCI (aq). The resulting solid was collected by vacuum
filtration, washed with water and dried under vacuum overnight. Column
chromatography (silica gel, 96:3:1 dichloromethane:methanol:conc. ammonium
I S hydroxide) yielded a light purple solid 103 (41 mg, 9%), mp 84 °C;
HPLC: YMC
Pack Pro C18, 40:30:30 [ICHZPO4 (0.01M, pH 3.2): CH30H: CH3CN], 264 nm,
Rt 12.7 min, 97% purity);'H NMR (600 MHz, CDC13,55 °C) 8 7.98 (s,
1H), 7.18
(S, IH), 6.85 (d, J= 9 Hz, IH), 6.58 (s, IH), 4.89 (s, 1H), 4.58-4.62 (m, 1H),
3.87
(s, 3H), 3.25 (t, J= 6.6 Hz, 2H), 3.05 (s, 3H), 2.94 (d, J= 11.4 Hz, 2H), 2.31
(s,
3H), 2.15 (S, 2H), 1.86 (dq, J= 12, 4.2 Hz, 3H), 1.57-1.78 (m, 8H), 1.15 -
1.30
(m, 4H), 1.00 (dq, J = I I .4, 3 Hz, 2H); MS (ESI): m/z 476 (37.7), 474 (M+H,
100), 410 (1.4).
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EXAMPLE 8
Synthesis of 6-Clzloro-N (3-chloro-4-rnethoxy phenyl)-N'-(1 propyl-butyl)-
~1,3,SJtriazine-2,4-diatnine (104)
OCH3
OCH3 CI
CI ~ I
NHZ HN
H N ~--N
-N N
~~-CI
~ =
~-CI N
N >
~N NaOH (aq), H20 HN
CI acetone, reflux,
101 3 hr, NZ atm
104
OCH3
CI
H /~
~N~N-CH3 /
CH3 ~/ HN
>--N CH3
NaOH (aq), HzO N~ ~~-N
1,4-dioxane ~-=N
reflux, 2 hr, N~ atm HN
N
GH3
105
To a sample of 101 (0.3062 g, 1.0 mmol) dissolved in acetone (4
mL) was added a solution of 4-heptylamine (0.15 mL, 1.0 mmol) in acetone (1
mL) followed by addition of a NaOH solution (0.0410 g, 1 mmol dissolved in 1
mL of HBO). The reaction mixture was allowed to stir at 30-SO°C for
about about
3 hours. The reaction mixture was then poured over crushed ice and neutralized
with 10% HCI (aq) and 5% NaOH (aq). The resulting solid was collected by
vacuum filtration, washed with water and dried overnight under vacuum to
afford
104 (0.363 g, 94% recovery).
EXAMPLE 9
Synthesis of N (3-Chloro-4-rzzethoxy pherzyl)-N'-methyl-N'-(1 anethyl
piperidiza-
4 yl)-N"-(1 propyl-bzrtyl)-~1,3,SJtriazine-2,4,6-triamine (105)
To a sample of 104 (0.363 g, 1.0 mmol) dissolved in 1,4-dioxane
(6 mL) was added a solution of N-methyl-4(methylamino)piperidine (0.15 mL,
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1.0 mmol) in acetone (1 mL) followed by addition of a NaOH solution (0.0414 g,
1.0 mmol dissolved in 1 mL of H20). The reaction mixture was allowed to stir
at
about 80°C for about about 2 hours. The reaction mixture was poured
over
crushed ice and neutralized with 10% HCl (aq). The resulting solid was
collected
by vacuum filtration, washed with water and dried under vacuum overnight.
Column chromatography (silica gel, 96:3:1 dichloromethane: methanol: conc.
ammonium hydroxide) yielded light purple solid 105 (97 mg, 20%), mp 249
°C.
HPLC: YMC Paclc Pro C18, 40:30:30 [I~HZP04 (O.OIM, pH 3.2): CH30H:
CH;CN], 264 nm, Rt 14.4 min, 98% purity; MS (ESI): n~lz 476 (M+H, 100),
412 (2.9), 366 (2.8), 239 (1.9).
EXAMPLE 10
Synthesis of N (3-Chloro-4-methoxy phenyl)-N'-isopropyl-N"-methyl-N"-(1-
methyl pipe~idin-4 yl)-~1,3,5Jtriazine-2,4,6-t~iamine (106)
OCH3
CI 1. isopropylamine, DIEA, CH3CN,
~ 1,4-dioxane, rt, overnight, Nz
atm
s +
H ~
HN Z' ~N~N-CH3
~J
~ CH
N 3
N ~>--CIpIEA, CH3CN, 1,4-dioxane, rt,
N t
i
ht
N
Z a
C m
g
,
overn
101 106 10'7
To a sample of 101 (0.6157 g, 2.0 mmol) dissolved in anhydrous
1,4-dioxane (15 mL) was added a solution of isopropylamine (0.17 mL, 2.0
mmol) in anhydrous acetonitrile (1 mL) followed by addition of a N,N
diisopropylethylamine (DIEA) (0.38 mL, 2.2 mmol) in anhydrous acetonitrile (1
mL). The reaction mixture was allowed to stir at room temperature overnight
under nitrogen. To this mixture was added DIEA (0.38 mL, 2.2 mmol) in
anhydrous acetonitrile (1 mL) followed by addition of N methyl-
4(methylamino)piperidine (0.29 mL, 2.0 mmol) in anhydrous acetonitrile (1 mL).
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The reaction mixture was allowed to stir at reflex overnight under nitrogen.
The
reaction mixture was extracted 3 times with ethyl acetate. The combined
organic
layers were washed one time with brine solution and dried over anhydrous
potassium carbonate. The organic layer was concentrated on a rotary evaporator
and allowed to dry overnight under vacuum. Column chromatography (silica gel,
93:6:1 CH2C12: CH30H: conc. NH40H) yielded light brown solid 106 (271 mg,
32%); TLC (silica gel, 93:6:1 CHZCIz: CH30H: conc. NH4OH), Rf 0.28; HPLC:
Inertsil ODS-3V C18, 40:30:30 [KH2P04 (O.OIM, pH 3.2): CH3OH: CH3CN],
264 nm, Rt 4.4 min, 84.8% purity; MS (ESI): n~/z 422 (26), 420 (M+H, 71.2),
378
(4.2), 231 (100), 211 (40.4), 118 (5.4).
EXAMPLE I1
Synthesis of N'-(3-ehloro-4-nZethoxy phenyl)-N~-isopropyl-Nb-methyl-N6-
pipe]°idin-4 yl-1,3,5-triazine-2,4,6-triamine (107)
Compound 107 was isolated (0.159 g) as a by-product via column
chromatography (silica gel, 93:6:1 CH2CI2: CH30H: conc. NH40H); mp 129
°C;
TLC (silica gel, 93:6: I CHZCl2: CH30H: conc. NH40H), Rf 0.14; HPLC: Inertsil
ODS-3V C18, 40:30:30 [I~HZP04 (O.OIM, pH 3.2) :CH30H: CH3CN~, 264 nm,
Rt 4.4 min, 93.5% purity; MS (ESI): mlz 408 (17.2), 406 (M+H, 46.6), 375
(18.5), 245 (11.9), 224 (100), 204 (13.4).
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EXAMPLE 12
Synthesis of 5-~4-(3-Chlo>'o-4-Inethoxy phenylami>zo)-6-methyl-(I->7lethyl-
~7lpG'1"IG113?-~-yl)-a79?1190~-~I,.3,JJt1"IaZl3?-2-ylaY121120~ pentalz-1-of
(1~~)
OCH3
CI
1. HZN\/°~f~OH
NN~ DIEA, CH3CN, 1,4-Dioxane, rt
N~N~--CI - °vernight, NZ atm
>=N H
CI ~' N~N-DHs
C H3
DIEA, CH3CN, reflex, overnight,
N~ atm
101 108 109
To a sample of 101 (1.5046 g, 5.0 mmol) dissolved in anhydrous
1,4-dioxane (30 mL) was added a solution of 5-amino-1-pentanol (0.5067 g, 5.0
mmol) in anhydrous acetonitrile (12 mL) followed by addition of N,N
diisopropylethylamine (DIEA) (0.95 mL, 5.5 mmol) in anhydrous acetonitrile (2
mL). The reaction mixture was allowed to stir at room temperature overnight
under nitrogen. To the reaction mixture was added DIEA (0.95 mL, 5.5 mmol) in
anhydrous acetonitrile (1 mL) . followed by addition of N methyl-
4(methylamino)piperidine (0.73 mL, 5.0 mmol) in anhydrous acetonitrile (1 mL).
The reaction mixture was allowed to stir at reflex overnight under nitrogen.
The
reaction mixture was extracted 3 times with ethyl acetate. The combined
organic
layers were washed one time with brine solution and dried over anhydrous
potassium carbonate. The organic layer was concentrated on a rotary evaporator
and allowed to dry overnight under vacuum. Column chromatography (silica gel,
90:9:1 CHaCl2: CH30H: cone. NH40H) yielded light brown solid 108 (300 mg,
13%); TLC (silica gel, 90:9:1 CH2ClZ: CH3OH: cone. NH40H), Rf 0.22; HPLC:
YMC Paclc Pro C18, 40:30:30 [KHZP04 (O.OIM, pH 3.2): CH30H: CH3CN], 264
nm, Rt 3.5 min, 74.8% purity; MS (ESI): rrllz 466 (24.2), 464 (M+H, 71.5), 378
(5.2), 253 (4.5), 244 (20.5), 233 (100), 216 (33.3), 196 (14.6), 118 (5.1).
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EXAMPLE 13
Synthesis of 5-(4-(3-chloro-4-methoxy plaenylamino)-6-(methyl piperidin-4 yl-
anaino)-1,3,5-triazin-2 ylam.inoJ pentan-I-of (109)
Compound 109 was isolated as a by-product (0.820 g) via column
chromatography (silica gel, 90:9:1 CHZC12: CH30H: conc. NH40H), mp 101
°C;
TLC (silica gel, 90:9:1 CH2ClZ: CH30H: conc. NH40H), Rf0.08; HPLC: Inertsil
ODS-3V C18, 40:30:30 [KH2PO4 (O.OIM, pH 3.2): CH30H: CH3CN], 264 nm,
Rt 3.6 min, 95.3°f° purity; MS (ESI): m/z 452 (13), 450 (M+H,
35.6), 419 (3.9),
267 (5.1), 246 (100), 226 (21.3), 209 (23.6), 118 (1.1).
EXAMPLE 14
Synthesis of N Butyl-6-chloro-N'-(3-chloro-4-rnethoxy phenyl)-N propyl-
~1,3,SJtriazine-2,4-diarnine (110)
IS
OCH3
OCH3 CI
CI
T t-i HN
HN. ~N~ ~N
N ~~--CI ~N CI
=N NaOH (aq), acetone, /N
CI 30-35°C, 3 hrs, NZ atm
101 110
OCH3 OCH3
CI \ .
H /'~
~N~N-CH3
cH3 ~--~ HN .1.
N~N NCH3
NaOH (aq), 1,4-dioxane a
reflux, 2 hrs, NZ atm ~N
~N~
CH3 ~H
111 112
To a sample of 101 (1.5334 g, 5.0 mmol) dissolved in acetone (20
mL) was added a solution of N propyl-butylamine (0.77 mL, 5.0 mmol) in
acetone (1 mL) followed by the addition of NaOH (2.0 mL, 2.5 N, 5.0 mmol).
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The reaction mixture was allowed to stir at 30-35°C for about 3
hours under
nitrogen. The reaction mixture was extracted 3 times with dichloromethane; the
combined organic layers were washed with brine solution and dried over
potassium carbonate. The sample was concentrated on a rotary evaporator and
the resulting oil was dried overnight under vacuum. Column chromatography
(silica gel, 96:3:1 dichloromethane: methanol: conc. ammonium hydroxide)
yielded light brown solid 110 (1.4 g, 77% recovery).
EXAMPLE 15
Synthesis of N Butyl-N'-(3-chloro-4-methoxy phenyl)-N"-methyl-N"-(I-methyl-
piperidin-4 yl)-Npropyl-~1,3,SJtriazine-2,4,6-triamine (111)
To a sample of 110 (1.323 g, 3.4 mmol) dissolved in 1,4-dioxane
(25 mL) was added a solution of N methyl-4(methylamino)piperidine (0.4 mL,
3.4 mmol) in 1,4-dioxane (1 mL) followed by the addition of NaOH (1.4 mL, 2.5
N, 3.4 mmol). The reaction mixture was allowed to stir at reflux for about 2
hours
under nitrogen. The reaction mixture was extracted 3 times with
dichloromethane; the combined organic layers were washed with brine and dried
over potassium carbonate. The sample was concentrated on the rotary evaporator
and the resulting solid was dried overnight under vacuum. Column
chromatography (silica gel, 90:9:1 dichloromethane: methanol: cone. ammonium
hydroxide) yielded light brown solid compound 111 (527 mg, 33%), mp 68
°C;
TLC (silica gel, 90:9:1 CH2Clz: CH30H: conc. NH40H), Rf 0.46; HPLC: OI~S
3 V C I 8, 40:30:30 [I~HzP04 (0.01 M, pH 3.2): CH30H: CH3CN], 264 nm, Rt 41.6
min, 90.8% purity); MS (ESI): m/z 476 (M+H, 28.5), 261 (20.2), 260 (52.8), 259
(100), 239 (18.6), 239 (50.6).
EXAMPLE 16
Synthesis of N~-Butyl-lVa-(3-chloro-4-methoxy phenyl)-IV6-methyl-1V6 piperidin-
4-
yl-N~ propyl-l, 3, 5-triazine-2, 4, 6-triamine (112)
Compound 112 was isolated as a by-product via column
chromatography, an oil (0.112 g); TLC (silica gel, 90:9:1 CH2Cla: CH30H: conc.
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NH40H), Rf 0.23; HPLC: ODS-3V C18, 40:30:30 [KH2P04 (0.01M, pH 3.2):
CH30H: CH3CN], 265 nm, Rt 41.4 min, 97.8% purity); MS (ESI): m/z 464
(11.6), 462 (M+H, 28.9), 431 (15.6), 273 (12.7), 253 (58.8), 252 (100), 232
(25.8), 157 (14.5).
EXAMPLE 17
Synthesis of 2,4-Dichloro-6-cyclohexylrnethoxy-~1,3,SJtriazine (113)
F
c1 H~~ c1 H2N \ / ocH3
~N ~ ~N
N -N C) toluene, 18-crown-6 ~CI NaOH (aq), acetone,
CI ICZC03, reflux, NZ atm O reflux 3 hrs, Nz atm
113
OCH3 OCH3
F \ F
I /
HN HN
N ~>--CI "E' N ~NH
N ~ N ~ ~ F
OCH3
114 115 ~ 116
HEN
NaOH (aq), 1,4-dioxane
acetone, reflux, 2 hrs,
NZ atm
To cyanuric chloride (3.76 g, 20.0 mmol) dissolved in toluene (20
mL) was added potassium bicarbonate (2.80 g, 20.0 mmol) and 18-crown-6
(0.1614 g, 0.6 mmol) followed by dropwise addition of cyclohexylmethanol (2.5
mL, 20 mmol) in 15 mL of toluene (15 mL). The reaction mixture was allowed
to stir at reflux for about 18 hours under nitrogen. The reaction mixture was
passed through a plug of Celite and concentrated using a rotary evaporator and
dried over night under vacuum to give 113 as an oil (5.212 g, 99% recovery).
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EXAMPLE 18
Synthesis of (4-Chloro-6-cyclohexylmethoxy-~1,3,SJtriazir~-2 y1)-(3 fluoro-4-
methoxy phenyl)-amine (114)
To a sample of 113 (1.011 g, 3.8 mmol) dissolved in acetone (20
mL) was added a solution of 3-fluoro p-anisidine (0.541 g, 3.8 mmol) in
acetone
(10 mL) followed by addition ofNaOH (1.52 mL, 2.5 N, 3.8 mmol) and water (3
mL). The reaction mixture was allowed to stir at reflux for about 3 hours
under
nitrogen. The reaction mixture was extracted 3 times with dichloromethane; the
combined organic layers were washed with brine solution and dried over
potassium carbonate. The sample was concentrated on a rotary evaporator and
the resulting oil was dried overnight under vacuum. Column chromatography
(silica gel, 70:30 hexanes: ethyl acetate) yielded light yellow solid compound
114
(0.581 g, 42%), mp 98 °C; TLC (silica gel, 30: 70 ethyl acetate:
hexanes), Rf
0.36; MS (ESI): m/z 369 (39.1), 368 (22.1), 367 (M+H, 100), 273 (3.2), 271
(10.7).
EXAMPLE 19
Synthesis of 6-Cyclohexylmethoxy-N,N'-bis-(3 fluoro-4-methoxy phenyl)-1,3,5-
triazine-2, 4-dianZine (11 S)
Compound 115 was obtained as a by-product (0.159 g) via column
chromatography (silica gel, 70:30 hexanes: ethyl acetate), mp 181 °C;
TLC
(silica gel, 30: 70 ethyl acetate: hexanes), Rf 0.17; MS (ESI): m/z 472 (M+H,
100), 261 (1.5).
EXAMPLE 20
Synthesis of 6-Cyclohexylmethoxy-N (1-ethyl pyrrolidin-2 ylmethyl)-N'-(3-
fluoro-4-methoxy pl~e~yl)-~1,3,SJtriazir~e-2,4-diamine (116)
To a sample of 114 (0.3004 g, 0.82 mmol) dissolved in 1,4
dioxane (IS mL) was added a solution of 2-(aminomethyl)-1-ethylpyrrolidine
(0.12 mL, 0.82 mmol) in acetone (1 mL) followed by the addition of NaOH (0.33
mL, 2.5 N, 0.82 mmol) and water (1 mL). The reaction mixture was allowed to
stir at reflux for about 2 hours under nitrogen. The reaction mixture was
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extracted 3 times with dichloromethane; the combined organic layers were
washed with brine and dried over potassium carbonate. The sample was
concentrated on the rotary evaporator and the resulting solid was dried
overnight
under vacuum. Column chromatography (silica gel, 93:6:1 dichloromethane:
methanol: conc. ammonium hydroxide) yielded a light yellow solid, compound
116 (226 mg, 60%), mp 59 °C; HPLC: Inertsil ODS-3V C18, 40:30:30
[KHaP04
(0.01M, pH 3.2): CH30H: CH3CN], 264 nm, Rt 10.5 min, 100% purity; IH NMR
(600 MHz, CDCI3, 55 °C) 8 7.65 (broad resonance, rotamers, 1H), 7.07
(br d, J=
7.8 Hz, 1 H), 6.90 (t, J= 9 Hz, 1 H) 6.84 (broad resonance, rotamers, 1H),
4.12 (s,
2H), 3.88 (S, 3H), 1.02 (s, 1 H), 2.26 (apt sextet, J = 6.6 Hz, 1H), 2.19 (q,
J =
9Hz, 1 H), I .16-1.92 (m, 1 OH), 1.57 (s, 2H), 1.17-1.32 (m, 3H), 1.05 -1.11
(m,
4H); MS (ESI): m/z 459 (M+H, 100), 363 (40.7), 223 (16.1), 202 (4.4), 138
( I .2).
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EXAMPLE 21
Synthesis of (4-Chloro-6-cyclohexylrnethoxy-(1,3,SJtriazin-2 yl)-(3-chloro-4-
n~ethoxy phenyl)-amine (117)
OCFI3 OCH3
CI GI ~ GI
~N H2N \ / OcH3 ~ a I i
>=NCI NaOH (aq), acetone, HN~N -I- HN~N
O reflux. 3 hrs, N~ atm N ~~CI N ~~--NH
O N O N I \ C
I
O
113 117 ~ 118 G
Ha
OCH3
CI
HN
>---N GH3
N~ ~~--N NaOH (aq), 1,4-dioxane
N acetone, reflux, 2 hrs,
O ~ NZ atm
~CH3
119
To a sample of compound 113 (1.012 g, 3.8 mmol) dissolved in
acetone (20 mL) was added a solution of 3-chloro p-anisidine (0.605 g, 3.8
mmol) in acetone (10 mL) followed by addition of NaOH (1.52 mL, 2.5 N, 3.8
mmol) and water (3 mL). The reaction mixture was allowed to stir at reflux for
'about 3 hours under nitrogen. The reaction mixture was extracted 3 times with
dichloromethane; the combined organic layers were washed with brine and dried
over potassium carbonate. The sample was concentrated on a rotary evaporator
and the resulting oil was dried overnight under vacuum. Column
chromatography (silica gel, 70:30 hexanes: ethyl acetate) yielded a light
peach
colored solid, compound 117 (0.547 g, 38%), mp 114 °C; TLC (silica gel,
30: 70
ethyl acetate: hexanes), Rf 0.44; MS (ESI): m/z 385 (74.3), 384, (22.9), 383
(M+H, 100), 287 (8.3).
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EXAMPLE 22
Syrrthe,s~is of N,N'-Bis-(3-clzloro-4-nzethoxy phefzyl)-6-cyclohexylrnethoxy-
1,3,5-
triazine-2,~-dianzizze (118)
Compound 118 was obtained as a by-product (0.178 g) via column
chromatography (silica gel, 70:30 hexanes: ethyl acetate), mp 188 °C;
TLC (silica
gel, 30: 70 ethyl acetate: hexanes), Rf 0.22; MS (ESI): zn/z 504 (M+H, 100),
379
(I), 338 (1.3).
EXAMPLE 23
Synthesis of N (3-Chloro-4-nzethoxy phezzyl)-6-cyclohexylmethoxy-N'-methyl-N'-
(1-methyl piperidin-4 yl)-(I, 3, SJtriazirze-2, 4-dia»zizze (119)
To a sample of 117 (0.3007 g, 0.78 mmol) dissolved in 1,4
dioxane (15 mL) was added a solution of 2-(aminomethyl)-1-ethylpyrrolidine
(0.11 mL, 0.78 mmol) in acetone (I mL) followed by the addition of NaOH (0.31
mL, 2.5 N, 0.78 mmol) and water (1 mL). The reaction mixture was allowed to
stir at reflux for about 2 hours under nitrogen. The reaction mixture was
extracted 3 times with dichloromethane; the combined organic layers were
washed with brine solution and dried over potassium carbonate. The sample was
concentrated on the rotary evaporator and the resulting solid was dried
overnight
under vacuum. Column chromatography (silica gel, 93:6:1 dichloromethane:
methanol: conc. ammonium hydroxide) yielded light yellow solid compound 119
(I59 mg, 43%), mp 140 °C. HPLC: Inertsil ODS-3V C18, 40:30:30 [I~H2P04
(0.01 M, pH 3.2): CH30H: CH3CN], 264 nm, Rt I5.2 min, 99.7% purity; MS
(ESI): znlz475 (M+H, 64.1), 379 (49.5), 231 (48.6), 210 (100), 190 (3.2).
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EXAMPLE 24
Sy~athesis of 6-Chloj~o-N,N"-bis-(3-chloro-4-methoxy phenyl)-~1,3,SJtriazine-
2,4-
diaruine (120)
OCH3
OCH3 CI
CI ~
HN
c1
N
H N~N CI H2N \ / ocH3 N ~~--CI
_ ?=N
~N NaOH (aq), acetone, RT HN
CI 3 hrs, Nz atm a 120
101
CI
OCH3
OCH3
H /~
~N~N-CH3
CH V3
NaOH (aq), 1,4-dioxane
reflux, 2 hrs, N~ atm
To a sample of 101 (3.0556 g, 10.0 mmol) dissolved in acetone
(25 mL) was added a solution of 3-chloro p-anisidine (1.6050 g, 10.0 mmol) in
acetone (10 mL) followed by addition ofNaOH (4.0 mL, 2.5 N, 10.0 mmol). The
reaction mixture was allowed to stir at rt for about 3 hours under nitrogen.
The
reaction mixture was poured over crushed ice. The resulting solid was
collected
by vacuum filtration, washed with water and dried overnight under vacuum to
give compound 120 (4.06 g, 95%), mp 213 °C; HPLC: Inertsil ODS-3V C18,
40:30:30 [I~HaP04 (0.01 M, pH 3.2): CH30H: CH3CN], 264 nm, Rt 70.0 min,
97.1 % purity MS (ESI): m/z 427 (20.90), 426 (M+H, 99.6), 210 (100), 209
(22.2), 196 (55.3), 169 (25.4).
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EXAMPLE 25
Synthesis of N,N'-Bis-(3-chloro-4-methoxy ~herzyl)-N"-methyl-N"-(4-methyl-
cyelohexyl)-~l, 3, SJtriazine-2, 4, 6-triamine (121)
To a sample of compound 120 (1.5004 g, 3.5 mmol) dissolved in
1,4-dioxane (20 mL) was added a solution of N methyl-4(methylamino)-
piperidine (0.5 mL, 3.5 mmol) in 1,4-dioxane (1 mL) followed by the addition
of
NaOH (1.4 mL, 2.5 N, 3.5 mmol). The reaction mixture was allowed to stir at
reflex for about 2 hours under nitrogen. The reaction mixture was poured over
crushed ice and neutralized with 10% HCl (aq). The resulting solid was
collected
l0 by vacuum filtration, washed with water and dried overnight under vacuum.
Column chromatography (silica gel, 96:3:1 dichloromethane: methanol: cone.
ammonium hydroxide) yielded a purple solid, compound 121 (487 mg, 27%), mp
130 °C; HPLC: Inertsil ODS-3V C18, 40:30:30 [I~H2POø (0.01 M, pH 3.2):
CH30H: CH3CN], 264 nm, Rt 8.1 min, 96% purity;'H NMR (600 MHz, CDC13,
55 °C) 8 7.81-7.92 (broad resonance, 2H), 7.19-7.30 (broad resonance,
2H), 6.87
(d, J= 9 Hz, 2H), 6.72 (s, 2H), 4.60-4.65 (m, 1H), 3.88 (s, 6H), 3.05 (s, 3H),
2.95
(d, J = I 2 Hz, 2H), 2.32 (s, 3H), 2.19 (t, J = 1 I .4Hz, 2H), 1.89 (dq, J
12.6, 3.6
Hz, 2H), I .71 (apt d, J = 11.4Hz, 2H), 1.65 (s, 1 H); MS (ESI): m/z 519
(28.3),
518 (M+H, 42.1), 261 (71.9), 260 (100).
EXAMPLE 26
Syr~tlaesis of N,N'-Bis-(3-chloro-4-methoxy phenyl)-N"-cycloheptyl-
~1,3,SJtriazine-2,4,6-triamine (122)
OCH3 NHS
CI
T
HN
N
N ~~-CI NaOH (aq), acetone
=N reflex, 2 hrs, N~ atm
HN
OCH3 120
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To a sample of 120 (1.5004 g, 3.5 mmol) dissolved in acetone (20
mL) was added a solution of cycloheptylamine (0.4 mL, 3.5 mmol) in acetone (1
mL) followed by the addition of NaOH (1.4 mL, 2.5 N, 3.5 mmol). The reaction
mixture was allowed to stir at reflux for about 2 hours under nitrogen. The
reaction mixture was poured over crushed ice and neutralized with 10% HCl
(aq).
The resulting solid was collected by vacuum filtration, washed with water and
dried overnight under vacuum to give light purple solid compound 122 (1.5 g,
85%), mp 183 °C; HPLC: Inertsil ODS-3V C18, 40:30:30 [KH2PO4 (0.01 M,
pH
3.2): CH30H: CH3CN~, 264 nm, R~ 59 min, 96% purity; MS (ESI): m/z 503
(M+H, 29), 502 (100), 458 (24.2), 425 (17.9), 225 (5.7), 155 (11.3), 114
(27.6).
EXAMPLE 27
Synthesis of N (3-Bronco-4-methoxy phenyl)-N'-cyeloheptyl-N"-methyl-N"-(1-
methyl piperidin-4 yl)-~1,3,SJtriazine-2,4,6-triamine (1~3)
Br
_ OCH3
HZN ~ / ocH3
1.
C.1 DIEA, CH3CN, -10°C, rt, NZ atm
~N
N ~~--CI
~N 2. ~NHz
CI
DIEA, CH3CN, reflux, NZ atm
CH3 ~
~N~N-CH3
3. NH
123
DIEA, CH3CN, reflux, NZ atm
To cyanuric chloride (0.184 g, 1.0 mmol) dissolved in acetonitrile
(3 mL) stirring at about -10° C, was added a solution of 3-bromo p-
anisidine
(0.2019 g, 1.0 mmol) in acetonitrile followed by the addition of N,N
diisopropylethylamine (DIEA) (0.17 mL, 1.0 mmol) in acetonitrile. The reaction
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mixture was allowed to stir at about -10° C for 1 hour under nitrogen.
The
reaction mixture was then warmed to room temperature and allowed to stir at
room temperature for another hour under nitrogen. To the reaction mixture was
added a solution of cycloheptylamine (0.13 mL, 1.0 mmol) in acetonitrile
followed by addition of DIEA (0.17 mL, 1.0 mmol). The reaction mixture was
allowed to stir at reflux overnight under nitrogen. To the reaction mixture
was
added N methyl-4(methylamino)piperidine (0.13 mL, 1.0 mmol) in acetonitrile
followed by the addition of DIEA (0.17 mL, 1.0 mmol). The reaction mixture
was allowed to stir at reflux for overnight under nitrogen. The reaction
mixture
was extracted 3 times with ethyl acetate; the combined organic layers were
washed with brine solution and dried over potassium carbonate. The sample was
concentrated on the rotary evaporator and the resulting solid was dried
overnight
under vacuum. Column chromatography (silica gel, 90:9:1 methylene chloride:
methanol: conc. ammonium hydroxide yielded 0.029 g (6%)of 123. 'H NMR
(400 MHz, GDC13) ~ 7.97- 8.19 (broad resonance, 1H), 7.12 (broad resonance,
IH), 6.78-6.80(m, 2H), 4.82 (br s, IH), 4.58 (br s, 1H), 3.92 (br s, 1H), 3.84
(s,
3H), 2.90-2.98 (m, SH), 2.29 (s, 3H), 2.17 (broad resonance, 2H), 1.99-2.24
(broad resonance, 4H), 1.72-1.85 (m, 3H), 1.42-1.62 (m, 11H); MS (ESI): m/z
520 (100), 518 (93.9), 458 (10.4), 424 (20.8), 422 (21.1), 261 (67.5), 260
(63.4);
213 (13.9), 212 (13.6).
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EXAMPLE 28
Synthesis of 6-Chloro-N cyelohexylrnethyl-N'-(3 fluoro-4-methoxy phenyl)-
~1,3,SJt~°iazine-2,4-diamine (125)
OCH3
F
HN ~
N H2N
N ~>--CI
=N NaOH (aq), HZO, acetone,
CI reflux, 4 hr, Na atm
124
OCH3
F w HaN V
0
HN
N ~CI NaOH (aq), HaO,1,4-dioxane,
=-N acetone reflux, 2 hr, Na atm
HN
125 126
To a sample of 124 (40.02 g, 138.4 mmol, prepared as indicated
herein) dissolved in acetone (300 mL) was added a solution of
cyclohexanemethylamine (18.0 mL, 138.4 mmol) in acetone (30 mL) followed by
addition of NaOH (55.4 mL, 2.5 N, 138.4 mmol) and 130 mL of water. The
reaction mixture was allowed to stir at reflux for about 3 hours. The reaction
mixture was then poured over crushed ice and neutralized with 10% HCI (aq) and
10% NaOH (aq). The resulting solid was collected by vacuum filtration, washed
with water and dried overnight under vacuum. Recrystallization from ethyl
acetate yielded a light yellow solid, compound 125 (32.93 g, 65%), mp 156
°C;
HPLC: Inertsil ODS-3V C18, 40:10:50 [I~H2P04 (O.OlM, pH 3.2): CH3OH:
CH3CN], 264 nm, Rt 47.9 min, 92 % purity; MS (ESI): m/z 366 (M+H, 100).
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EXAMPLE 29
Synthesis of N Cyclohexylmethyl-N'-(1-ethyl pyrrolidizz-2 ylmethyl)-N"-(3-
flZtoro-4-metlzoxy p7zenyl)-(1,3,5Jtriazirte-2,4,6-triamine (126)
To a sample of 125 (10.02 g, 27.3 mmol) dissolved in 1,4-dioxane
(150 mL) was added a solution of 2-(aminomethyl)-1-ethylpyrrolidine (4.0 mL,
27.3 mmol) in acetone (10 mL) followed by addition of NaOH (11 mL, 2.5 N,
27.3 mmol) and 27 mL of water. The reaction mixture was allowed to stir at
reflux for about 2 hours. The reaction mixture was extracted 3 times with
dichloromethane; the combined organic layers were washed with brine and dried
over potassium carbonate. The sample was concentrated on the rotary evaporator
and the resulting solid was dried overnight under vacuum. Column
chromatography (silica gel, 93:6:1 dichloromethane: methanol: conc. ammonium
hydroxide) yielded a light yellow solid, compound 126 (7.014 g, 56%), mp 72
°C; HPLC: Inertsil ODS-3V C18, 40:30:30 [ICH2PO4 (O.OlM, pH 3.2):
CH30H:
CH3CN], 264 nm, R~ 8.5 min, 93.4 % purity; MS (ESI): m/z 458 (M+H, 37.3), .
362 (4), 250 (100), 230 (15.3), 229 (44.1).
EXAMPLE 30
Synthesis of N Cyclolzeptyl-N'-(3 fluoro-4-methoxy phenyl)-6 pyrrolidin-1-yl-
~1,3,5Jtriazine-2,4-dianzine (12~)
OCH3
H
N
HN
N
N ~>-C~ NaOH (aq), H20
~N THF, retlux
HN 2 hr, N~ atm
127 128
To a sample of compound 127 (13.24 g, 36.2 mmol, prepared as
indicated herein) dissolved in THF (150 mL) was added a solution of
pyrrolidine
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(3.0 mL, 36.2 mmol) in THF (10 mL) followed by addition of NaOH (14.5 mL,
2.5 N, 36.2 mmol) and 36 mL of water. The reaction mixture was allowed to stir
at reflux for about 2.5 hours. The reaction mixture was extracted 3 times with
dichloromethane; the combined organic layers were washed with brine and dried
over potassium carbonate. The sample was concentrated on the rotary evaporator
and the resulting solid was dried overnight under vacuum. Column
chromatography (silica gel, 98: 2 dichloromethane: methanol) yielded ~ light
yellow solid 128 (3.36 g, 23%), mp 79 °C; HPLC: Inertsil ODS-3V C18,
40:10:50 [KHzPO4 (O.OIM, pH 3.2): CH30H: CH3CN], 264 nm, Rt 24.5 min,
95.5% purity;'H NMR (600 MHz, CDCI3, 55 °C) 8 7.77 (broad resonance,
1H),
7.01-7.03 (m, I H), 6.86 (t, J = 9 Hz, I H), 6.62 (s, 1 H), 4.80 (s, 1H), 4.02-
4.06
(m, IH), 3.85 (s, 3H), 3.54 (s, 4 H), 1.99-2.03 (m, 2H), 1.91-1.93 (m, 3H),
1.47-
1.66 (m, 11H); MS (ESI): m/z 402 (30.7), 401 (M+H, 100).
EXAMPLE 31
Synthesis of (4,6-1)ichloro-~1,3,SJtricrzih-2 yl)-(3-fluoro-4-methoxJr
phe~zyl)-
anzine (124)
OCH3
F \
F I / NHz
C~N HzN \ / ccH3 HN~N
N ~~CI N ~~CI
=N NaOH (aq), N NaOH (aq), HZO
CI acetone, 0-5 C, CI acetone, reflux,
124 3 hr, Nz atm
OCH3
OCH3 F \
F \
(
CH3CH~NH~ HN
HN ~N H
~N NaOH (aq), Hz0 N ~~-N
N ~~-CI THF, reflux ~=N
=N 2 hr, NZ atm HN
HN
127 129
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To cyanuric chloride (28.84 g, 156.0 mmol) dissolved in acetone
(200 mL) stirring at approximately 0-5° C, was added a solution of 3-
fluoro p-
anisidine (22.16 g, 156.0 mmol) in acetone (200 mL) followed by the addition
of
NaOH (63 mL, 2.5 N, 156.0 mmol). The reaction mixture was allowed to stir at
approximately 0-5° C for about 2 hours. The reaction mixture was then
poured
over crushed ice and neutralized with 10% HCI (aq) and S% NaOH (aq). The
resulting solid was collected by vacuum filtration, washed with water and
dried
overnight under vacuum. Column chromatography (silica gel, 70:30 hexane:
ethyl acetate) yielded light yellow solid compound 124 (29.6 g, 66%); mp 134
°C; HPLC: Inertsil ODS-3V C18, 40:30:30 [I~HzP04 (0.01M, pH 3.2):
CH30H:
CH3CN], 264 nm, Rt 20.3 min, 97.7 % purity.
EXAMPLE 32
Synthesis of 6-Chloro-N cycloheheptyl-N'-(3 fluoro-4-methoxy phertyl)-
~1,3,SJtriazifze-2,4-diamine (127)
To a sample of 124 (10.00 g, 34.6 mmol) dissolved in acetone
(150 mL) was added a solution of cycloheptylamine (4.4 mL, 34.6 mmol) in
acetone (20 mL) followed by addition of NaOH (13.8 mL, 2.5 N, 34.6 mmol) and
35 mL of water. The reaction mixture was allowed to stir at reflux for about 3
hours. The reaction mixture was extracted 3 times with dichloromethane; the
combined organic layers were washed with brine and dried over potassium
carbonate. The sample was concentrated on the rotary evaporator and the
resulting solid was dried overnight under vacuum affording 127 (12.4 g, 98%
recovery); mp 145 °C; HPLC: Inertsil ODS-3V C18, 40:30:30 [KHZPO4
(0.01M,
pH 3.2): CH30H: CH3CN], 264 nm, Rt 104.8 min, 97.3 % purity; 1H NMR (600
MHz, CDC13, 55 °C) 8 7.50-7.64 (m, I H), 7.02-7.03 (br resonance, 2H),
6.90 (t,
,l = 8.9 Hz, 1H), 5.35-5.41 (br resonance, 1H), 3.99 (br s, 1H), 4.12
(rotamer),
3.87 (s, 3H), 2.01 (br s, 2H), 1.42-1.67 (m, I 1H).
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EXAMPLE 33
Synthesis of N Cycloheptyl-N'-ethyl-N"-(3-fluoro-4-methoxy phenyl)-
(1,3,5Jtriazi~e-2,4-diamine (129)
To 127 (I I .00 g, 30 mmol) dissolved in THF (150 mL) was added
a solution of ethylamine hydrochloride (2.43 mL, 30 mmol) in THF (20 mL)
followed by addition of NaOH (24 mL, 2.5 N, 60 mmol) and 30 mL of water.
The reaction mixture was allowed to stir at reflux for about 2 hours. The
reaction
mixture was extracted 3 times with dichloromethane; the combined organic
layers were washed with brine and dried over potassium carbonate. The sample
was concentrated on the rotary evaporator and the resulting solid was dried
overnight under vacuum. Column chromatography (silica gel, 98:2
dichloromethane: methanol) yielded a light yellow solid 129 (4.81 g, 43%), mp
84 °C; HPLC: Inertsil ODS-3V C18, 40:30:30 [KH2P04 (O.OlM, pH 3.2):
CH30H: CH3CN], 264 nm, Rt 30.7 min, 94.2 % purity; 'H NMR (600 MHz,
CDC13, 55°C) 8 7.69 (s, 1 H), 7.00 (br d, J= 7.0 Hz, 1H), 6.86 (t, J=
8.4 Hz, 1H),
6.64 (s, 1H), 4.79- 4.83 (br resonance, 2H), 4.01- 4.03 (m, 1H), 3.85 (s, 3H),
3.38-3.42 (m, 2H), 1.99-2.01 (m, 2H), 1.47-1.67 (m, 11H), 1.19 (t, J = 7.2 Hz,
3H); MS (ESI): m/z 376 (29.5), 375 (M+H, 100).
2p EXAMPLE 34
Synthesis of N Cycloheptyl-N'-(1-ethyl pyrrolidin-2 ylmethyl)-N"-(3 fluoro-4-
methoxy phenyl)-~1,3,SJtriazine-2,4,6-triamifze (130)
OCH3
OCH3 F
F
N
HZN~ HN
HN ~N H
NaOH (aq), HZO ~ \~N
N ~CI THF, reflux N
~N 2 hr, Nzatm HN
HN
127 130
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To 127 (5.009 g, 13.7 mmol) dissolved in THF (80 mL) was added
a solution of 2-(aminomethyl)-1-ethylpyrrolidine (2.0 mL, 13.7 mmol) in THF
(10 mL) followed by addition of NaOH (5.5 mL, 2.5 N, 13.7 mmol) and 13 mL
of water. The reaction mixture was allowed to stir at reflux for about 2 hours
under NZ atm. The reaction mixture was extracted 3 times with dichloromethane;
the combined organic layers were washed with brine and dried over potassium
carbonate. The sample was concentrated on the rotary evaporator and the
resulting solid was dried overnight under vacuum. Column chromatography
(silica gel, 90:9:1 dichloromethane: methanol: conc. ammonium hydroxide)
yielded a light yellow solid 130 (3.63 g, 58%), mp 76 °C; HPLC:
Inertsil ODS
3V C18, 40:30:30 [KHZP04 (0.01M, pH 3.2): CH30H: CH3CN], 264 nm, Rt 7.1
min, 97.1 % purity; MS (ESI): m/z 459 (16.5), 458 (M+H, 48.7), 362 (31.3), 250
(100), 230 (22.8), 229 (62.7), 222 (17.2), 202 (34).
EXAMPLE 35
Sy~zthesis of 2-(4-chloro-6-(3-chloro-4-methoxy phenylamino)-~1,3,SJtriazin-2-
ylaminoJ propane-1,3-diol (131) ,
CI
OMe
CI
OMe
HO HN
CH3HN N-CH3
H~ HO~NH~ N N
N ~N NaOH, acetone, water, HN~N~CI NaOH, 1,4-dioxane, water
I
CI~N~CI reflux, N2 ~ reflux, N~
OH OH
101 131 132
To 101 (0.6114 g, 2 mmol) dissolved in acetone (3 mL) was added
2-amino-propane-1,3-diol (0.1818g, 2 mmol) dissolved in acetone (1mL) and
water (1mL). Then water (1 mL) was added to the reaction mixture followed by
2.5 N NaOH (aq) (0.8 mL, 2 mmol). The reaction mixture was heated at reflux
for 3 h under a NZ atmosphere. The reaction mixture was diluted with ethyl
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acetate and washed 2 x brine. The organic layer was separated, dried over
anhydrous K2CO3, filtered, and concentrated under reduced pressure affording
0.634 g of a purple solid. The crude material was purified by silica gel flash
column chromatography eluting with 100% ethyl acetate affording a colorless
oil
131 (0.124 g, 18%); HPLC: Inertsil ODS-3V C18, 40:30:30 [KHzPO~ (0.01 M,
pH 3.2): CH30H: CH3CN], 264 nm, Rt 5.7 min, 83.3% purity; MS (ESI): m/z
360 (M+H, 100), 338 (10.7), 183 (10.3)
EXAMPLE 36
Synthesis of 2-(4-(3-chloro-4-rnethoxy pl2enylamino)-6-methyl-(I-methyl-
piperidifz-4 yl)-aminoJ-(1,3,SJtriazi~-2 ~rlaminoJ propane-1,3-diol (132)
To 131 (0.979 g, 0.271 mmol) dissolved in 3 mL 1,4-dioxane was
added methyl-4-(methylamino)piperidine (0.05 mL, 0.34 mmol) dissolved in 2
mL 1,4-dioxane followed by the addition of 2.5 N NaOH (aq) (0.11 mL, 0.275
mmol). The mixture was heated at reflex for 3 h 45 min, cooled to about room
temperature, and then concentrated under reduced pressure. The resulting
material was diluted with dichloromethane and filtered. The filtrate was then
concentrated affording 56.5 mg of material. The crude material was purified by
silica gel pipet column eluting with 100% methanol affording an white solid
132
(21.1 mg, 18%), mp 84 °C; MS (ESI): m/z 454 (34.7), 452 (M+H, 100), 422
(11.3), 248 (25.3), 247 (51.3), 157 (60.3), 129 (27.5).
EXAMPLE 37
Synthesis of N (1-benzyl piperidin-4 yl)-N'-(3-chloro-4-methoxy
phe~°eyl)-N"-
cycloheptyl-~1,3,SJ-2,4,6-triamine (134)
ci
home
home
HN \ H2N-CN-~ HJ
N~N Ph N N
HN'~N~c~ O~~'~ OH3CN, reflex, HN~N~NH
Na
N
134 ~ a
133
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To 133 (0.1252 g, 0.382 mmol, prepared as indicated herein)
dissolved in 3 mL acetonitrile was added N,N diisopropyl ethyl amine (DIEA)
(0.
07 mL, 0.382 mL) followed by 4-amino-I-benzylamine (0.07 mL, 0.382 mmol).
The mixture was refluxed overnight under a N2 atmosphere. The reaction
mixture was diluted with methylene chloride and washed with brine. The organic
layer was separated, dried over KzC03, filtered and concentrated under reduced
pressure to afford 0.159 g of material. The crude material was purified by
silica
gel flash column chromatography eluting with 96:3:1 methylene chloride:
methanol: conc. ammonium hydroxide and the collected fractions were dried over
potassium carbonate, filtered and then concentrated under reduced pressure to
afford 77 mg of product. A second column under similar conditions was
completed to afford an additional 30 mg of material for a combined product 134
(103 mg, 50%); HPLC: Inertsil ODS-3V C18, 40:30:30 [KH2PO4 (0.01 M, pH
3.2): CH3OH: CH3CN], 264 nm, Rt 13.7 min, 97.7% purity; MS (ESI): m/z 538
IS (15.4), 536 (38.2), 448 (19.3), 446 (49.3), 290 (41.4), 289 (84.6), 269
(100), 247
(4.4).
E~CAMPLE 38
Synthesis of Nz-(3-chlo~o-4-methoxy pherzyl)-Nø-cycloheptyl-lV6 pipe~idih-4 y1-
1,3,5-triazine-2,4,6-triamine (135)
c1
home
HN ~
N~N HCO~NH4, Pd/C
HN~N'~NH CH30H, reflux, Na
H
134 135
To 134 (0.0485 g, 0.0867 mmol) in 2 mL methanol was added
10% Pd/C (0.052 g) followed by ammonium formate (0.0646 g, 1.02 mmol). The
mixture was heated at reflux for about I .5 h under a Nz atmosphere. The
cooled
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reaction mixture was filtered by vacuum through Celite with a methylene
chloride rinsing, and the filtrate concentrated under reduced pressure to
afford 36
mg of material. The crude material was purified by silica gel flash
chromatography eluting with 90:9:1 methylene chloride: methanol: cons.
ammonium hydroxide, and the collected fractions were dried over potassium
carbonate, filtered and then concentrated under reduced pressure to afford a
solid
135 (20 mg, 51.8%), mp 167 °C; HPLC: Inertsil ODS-3V C18, 40:30:30
[KHZP04 (0.01 M, pH 3.2): CH30H: CH3CN], 264 nm, Rt 4.6 min, 52,1%
(another major peals at Ri 7.3 min, 46.9%); MS (ESI): m/z 448 (4.4), 446
(12.5),
412 (22.7), 386 (2.3), 265 (32.9), 248 (42.6), 244 (56.2), 228 (37.1), 227
(100),
207 (6.9).
EXAMPLE 39
Synthesis of N'-(3-chloro-4-rnethoxy phenyl)-Nø-cycloheptyl-1V6-(1-ethyl-
pyre~olidin-2 yl~nethyl)-1,3,x-triazine-2,4,6-tYiamihe (136)
CI c1
OMe OMe
H~ w ~ N w
N ~N ~NH~_ N~N
I
HN~N~CI DIEA, CH3CN, HN~N~NH
reflux, NZ
~N
133 136
To 133 (0.1257 g, 0.382 mmol, prepared as indicated herein)
dissolved in 3 mL acetonitrile was added DIEA (0.07 mL, 0.382 mL) followed by
2-(aminomethyl)-1-ethyl pyrrolidine (0.06 mL, 0.382 mmol). The mixture was
refluxed overnight under a Na atmosphere. The reaction mixture was diluted
with
methylene chloride and washed with brine. The organic layer was separated,
dried over KZC03, filtered and concentrated under reduced pressure to afford
0.143 g of material. The crude material was purified by silica gel flash
column
chromatography eluting with 96:3:1 methylene chloride: methanol: cons.
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ammonium hydroxide and the collected fractions dried over approximately 1:1
potassium carbonate/sodium sulfate, filtered and then concentrated under
reduced
pressure to afford 77 mg of product. A second column under similar conditions
was completed to afford an additional 30 mg of material for a combined 98 mg
(54%) of a yellow colored solid 136, mp 69-70 °C; HPLC: YMC Pack Pro
C18,
40:30:30 [KH2POa (0.01 M, pH 3.2): CH3OH: CH3CN], 264 nm, Rt 12.9 min,
96.5% purity; MS (ESI): m/z 476 (16.3), 474 (42.9), 260 (15), 259 (44.2), 258
(100), 238 (56), 216 (5.3), 210 (9.2).
EXAMPLE 40
Synthesis of 2-chloro-4-(4-cycloheptylamino-6-~rnethyl-(1-methyl piperidin-4
yl-
aminoJ-1,3,5-triazin-2 ylamino~ phenol (138)
ci ci
home / I OH
HN ~
N~N BBr3, CH2CI2, N~N
HN~N~N°CH3 HN~N~N'CH3
o cto n,
overnight, Np
N N
CH3 CH3
137 138
Under anhydrous conditions, 137 (0.1008 g, 0.21 mmol, prepared
as described herein) in a dry round bottomed flask was dissolved in anhydrous
methylene chloride (3 mL) under a NZ atmosphere about 0° C (ice/water
bath)
was added BBr3 (2.1 mL, 2.1 mmol, I M in methylene chloride) slowly by
syringe. The mixture was stirred for about 2 hours at about 0° C and
then
quenched with water (5 mL). After standing overnight at rt, the mixture was
diluted with ethyl acetate, water and 10% NaHC03 (aq), and the organic layer
was separated then washed with brine. The organic layer was then dried over
anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to
afford 0.648 g of material. The crude material was purified using silica gel
flash
column chromatography eluting with 100% methanol to afford of a white solid
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138 (7 mg, 7 %); HPLC: Inertsil ODS-3V C18, 40:30:30 [KHaP04 (0.01 M, pH
3.2): CH30H: CH3CN], 264 nm, Ri 4.9 min, 90.3% purity; 'H NMR (600 MHz,
CDCl3, 55°C) (all resonances are broad) 8 7.93 (s, 1H), 7.13 (s, 1H),
6.91- 6.92
(m 1H), 6.55 (s, 1H), 4.80 (s, 1H), 4.59 (s, 1H), 4.02 (s, 1H), 2.96 -3.0 (m,
SH),
2.32 (s, 3H), 2.13 (s, 2H), 2.03 (s, 2H), 1.86-1.88 (m, 2H), 1.53-1.67 (m,
12H);
MS (ESI): nalz 463 (12.4), 461 (27), 252 (59), 251 (100), 231 (32.3), 224 (1),
203
(9.8).
EXAMPLE 41
Synthesis ofN2-eycloheptyl-Nø-((S)-1-ethyl pyrrolidin-2 ylmethyl)-1V6-(3
fluoro-
4-methoxyphenyl)-1, 3, 5-triazine-2, 4, 6-triamine (139)
F
home
HN (°~~~
1. 3-fluoro-p-anisidine, CH3CN, iPraNEt,
N ~ N -10 to -20 C, 1 h; RT, 1 h
HN N NH
CI N CI 2. cycloheptane, iPrZNEt, RT, overnight l, H
3. S-(-)-2-aminomethyl-N-ethylpyrrolidine, iPrZNEt,
reflux, overnight
139
I S To a mixture of cyanuric chloride (0.368 g, 2 mmol) in CH3CN at
approximately -10 to -20°C was added 3-fluoro p-anisidine (0.28 g, 2
mmol) in
CH3CN followed by the addition of N,N diisopropylethylamine (DIEA) (0.35
mL, 2 mmol) and stirred for an hour. The reaction mixture was then allowed to
reach room temperature for an hour. The second step was continued without
further purification. Cycloheptylamine (0.25 mL, 2 mmol) and DIEA (0.35 mL,
2 mmol) were added and the reaction mixture was stirred overnight at rt.
°The
third step was also preceded without any further purification. S (-)-2-
aminomethyl-N ethyl pyrrolidine ( 0.29 mL, 2 mmol) and DIEA (0.35 mL, 2
mmol) were added and the reaction mixture was refluxed overnight. 'The
reaction
mixture was diluted with ethyl acetate and washed with brine. The organic
layer
was separated and dried over potassium carbonate, filtered, and concentrated
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under reduced pressure affording 0.920 g crude material. The crude material
was
purified by column chromatography to yield a white solid 139 (0.550 g, 60%),
mp 75-77 °C; HPLC: Inertsil ODS-3V C18, 40:30:30 [I~H~P04 (O.OlM, pH
3.2):
CH30H: CH3CN], 264 nm, R, 7.9 min, 95.9% purity; MS (ESI): m/z 458 (M+H,
I 00).
EXAMPLE 42
Synthesis of N2-cyclolzeptyl-N'~-((R)-1-ethyl pyrrolielin-2 ylmethyl)-IV6-(3
fluono-
4-methoxyplzenyl)-1,3,5-triazine-2,4,6-triamine (140)
F
OMe
HN ~
CI 1. 3-fluoro-p-anisidine, CH3CN, iPr~NEt, N~N
N~N -10 to -20 C, 1 h; RT, 1 h (
HN~N~NH
CI N CI 2. cycloheptane, iPr2NEt, RT, overnight
3. R-(+)-2-aminomethyl-N-ethylpyrrolidine, iPr2NEt,
reflux, overnight ~N
140
To a mixture of cyanuric chloride (0.368 g, 2 mmol) in CH3CN at
about -10 to -20°C was added 3-fluoro p-anisidine (0.28 g, 2 mmol) in
CH3CN
followed by the addition of N,N diisopropylethylamine (0.35 mL, 2 mmol) and
stirred for an hour. The reaction mixture was then allowed to reach room
temperature for an hour. Then cycloheptylamine (0.25 mL, 2 mmol) and DIEA
(0.35 mL, 2 mmol) were added and the reaction mixture was stirred overnight at
rt. To this reaction mixture R-(+)-2-aminomethyl-N ethyl pyrrolidine ( 0.29
mL,
2 mmol) and DIEA (0.35 mL, 2 mmol) were added and the reaction mixture was
refluxed overnight. The reaction mixture was diluted with ethyl acetate and
washed with brine. The organic layer was separated and dried over potassium
carbonate, filtered, and concentrated under reduced pressure affording 0.920 g
crude material. The crude material was purified by column chromatography to
yield a white solid 140 (0.500 g, 54.7%), mp 77-79 °C; HPLC: Inertsil
ODS-3V
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C18, 40:30:30 [KHZP04 (0.01 M, pH 3.2): CH30H: CH3CN], 264 nm, Rt 7.9
min, 74.3% purity; MS (ESI): rnlz 458 (M+H, 100).
EXAMPLE 43
Synthesis of NZ-cyclohexylmethyl-N~-((S)-1-ethyl pyrrolidin-2 ylmethyl)-1V6-(3-
flzaoro-4-methoxypheoyl)-1,3,5-triazine-2,4,6-tr'iamifze (141)
F
home
CI 1. 3-fluoro-p-anisidine, CH3CN, iPrZNEt, HN \ I
N ~ N -20 C, 1 h; RT, 1 h
~I N ~N
CI~N~CI 2. cyclohexylmethylamine, iPr2NEt, RT, overnight HN~N~NH
3. S-(-)-2-aminomethyl-N-ethylpyrrolidine, iPr2NEt,
reflux, overnight
~N
141
To a mixture of cyanuric chloride (0.368 g, 2 mmol) in CH3CN at
about -20°C was added 3-fluoro p-anisidine (0.28 g, 2 mmol) in CH3CN
followed
by the addition of N,N diisopropylethylamine (DIEA) (0.35 mL, 2 mmol) and
stirred for about 1 hour. The reaction mixture was then stirred at room
temperature for about 1 hour. Then, cyclohexylmethyl amine (0.26 mL, 2 mmol)
I S and DIEA (0.35 mL, 2 mmol) were added and the reaction mixture was stirred
overnight at RT. Then, S-(-)-2-aminomethyl-N ethyl pyrrolidine (0.29 mL, 2
mmol) and DIEA (0.35 mL, 2 mmol) were added and the reaction mixture was
refluxed overnight. The reaction mixture was diluted with ethyl acetate and
washed with brine. The organic layer was separated and dried over sodium
sulfate, filtered, and concentrated under reduced. The crude material was
purified
by column chromatography eluting with 96: 3: 1 methylene chloride: methanol:
cone. ammonium hydroxide to yield a white solid 141 (0.400 g, 43.7%), mp 68-
69 °C; HPLC: Inertsil ODS-3V C18, 40:30:30 [KHZPO4 (0.01 M, pH 3.2):
CH;OH: CH3CN], 264 nm, Itt 8.2 min, 97.1% purity; MS (ESI): m/z 458 (M+H,
100), 362 (2.8), 230 (85.4).
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EXAMPLE 44
Synthesis of N2-cyclohexylrrzethyl-N4-((R)-1-ethyl pyrrolidira-2 ylmethyl)-1V6-
(3-
fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4,6-tr~iamitze (142)
CI 1. 3-fluoro-p-anisidine, CH3CN, iPr2NEt,
-10 to -20 C, 1 h; RT, 1 h
I
CI N CI 2. cyclohexylmethylamine, iPrzNEt, RT, overnight
3. R-(+)-2-aminomethyl-N-ethylpyrrolidine, iPrZNEt,
reflux, overnight
142
To a mixture of cyanuric chloride (0.368 g, 2 mmol) in CH3CN at
about -20°C was added 3-fluoro p-anisidine (0.28 g, 2 mmol) in CH3CN
followed
by the addition of DIEA (0.35 mL, 2 mmol) and stirred for about 1 hour. The
reaction mixture was then stirred at room temperature for about 1 hour. Then,
cyclohexylmethyl amine (0.26 mL, 2 mmol) and DIEA (0.35 mL, 2 mmol) were
added and the reaction mixture was stirred overnight at room temperature.
Then,
R-(+)-2-aminomethyl-N ethyl pyrrolidine (0.29 mL, 2 mmol) and DIEA (0.35
mL, 2 mmol) were added and the reaction mixture was refluxed overnight. The
IS reaction mixture was diluted with ethyl acetate and washed with brine. The
organic layer was separated and dried over sodium sulfate, filtered, and
concentrated under reduced pressure. The crude material was purified by column
chromatography eluting with 96: f: 1 methylene chloride: methanol: cone.
ammonium hydroxide to give 142 (0.100 g, 10.9%), mp 66-67 °C; HPLC:
Inertsil
ODS-3V C18, 40:30:30 [KHaP04 (0.01 M, pH 3.2): CH3OH: CH3CN], 264 nm,
Rt 8.2 min, 96.7% purity; 'H NMR (600MHz, CDC13) 8 7.58-7.73 (broad
resonance, 1 H), 7.07-7.11 (broad resonance, 1H), 6.82 (t, J = 9 Hz, 1H), 5.49-
5.65 (broad resonance, I H), 4.96-5.13(broad resonance, 1H), 3.82 (s, 3H),
3.54-
3.70 (broad resonance, 1 H), 3.13-3.20 (br m, 4H), 2.81 (broad resonance, 1H),
2.54 (broad resonance, 1 H), 2.05-2.18 (m, 2H), 2.01 (s, 1H), 1.50-1.83 (br m,
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9H), 1.05-1.22 (m, SH), 0.91 (apt q, J = 11.4 Hz, 2H); MS (ESI): m/z 458
(M+H, 100), 362 (3.8), 230 (99.8), 216 (1), 182 (1.1).
EXAMPLE 45
Synthesis of (~4-cycloheptylamino-6-~((S)-1-ethyl pyrrolidin-2 ylmethyl)-
aminoJ-
1,3,5-triazi~-~ yl,~ phenyl-amino)-acetonitrile (143)
i
NC~N
CI 1. N-phenyl glycinonitrile, CH3CN, iPraNEt,
N~N -20 C, 1 h; RT, 1 h N~N
~ J~ J.
CI~N~CI 2, cycloheptylamine, iPrZNEt, RT, overnight HN N NH H
3. S-(-)-2-aminomethyl-N-ethylpyrrolidine, iPr2NEt,
reflux, overnight N
143
To a mixture of cyanuric chloride (0.368 g, 2 mmol) in CH3CN at
about -20°C was added N phenyl glycinonitrile (0.264 g, 2 mmol) in
CH3CN
followed by the addition of DIEA (0.35 mL, 2 mmol) and stirred for about 1
hour. The reaction mixture was then stirred at room temperature for about 1
hour. Then, cycloheptylamine (0.25 mL, 2 mmol) and DIEA (0.35 mL, 2 mmol)
were added and the reaction mixture was stirred overnight at rt. Then, S (-)-2-
aminomethyl-N ethyl pyrrolidine (0.29 mL, 2 mmol) and DIEA (0.35 mL, 2
mmol) were added and the reaction mixture was refluxed overnight. The reaction
mixture was diluted with ethyl acetate and washed with brine. The organic
layer
was separated and dried over sodium sulfate, filtered, and concentrated under
reduced pressure. The crude material was purified by column chromatography
eluting with 96: 3: I methylene chloride: methanol: conc. ammonium hydroxide
to yield 143, (0.300 g, 33°!°) mp 53-55 °C; HPLC:
Inertsil ODS-3V C18,
40:30:30 [KHZP04 (0.01 M, pH 3.2): CH30H: CH3CN], 264 nm, Rt 6.9 min,
94.1% purity; MS (ESI): ~a7/z 449 (M+H, 100), 381 (1.2), 353 (16.2), 226
(19.9),
225 (54.3), 212 (20.5), 177 (18.3), 164 (9.6).
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EXAMPLE 46
Sy>zthesis of (~4-cycloheptylamino-6-(((R)-1-ethyl pyrrolidin-2 ylrnethyl)-
ami>zoJ-
1,3,5-triazin-2 yl) phenyl-amino)-acetonitrile (144)
C) 1. N-phenyl glycinonitrile, CH3CN, iPr2NEt,
N~N -20 C, 1 h; RT, 1 h
~I
CI~N~CI 2. cycloheptylamine, iPraNEt, RT, overnight
3. R-(+)-2-aminomethyl-N-ethylpyrrolidine, iPr2NEt,
reflux, overnight
144
To a mixture of cyanuric chloride (0.368 g, 2 mmol) in CH3CN at
about -20°C was added N phenyl glycinonitrile (0.264 g, 2 mmol) in
CH3CN
followed by the addition of DIEA (0.35 mL, 2 mmol) and stirred for about 1
hour. The reaction mixture was then stirred at room temperature for about 1
hour. Then, cycloheptylamine (0.25 mL, 2 mmol) and DIEA (0.35 mL, 2 mmol)
were added and the reaction mixture was stirred overnight at rt. Then, R-(+)-2-
aminomethyl-N ethyl pyrrolidine (0.29 mL, 2 mmol) and DIEA (0.35 mL, 2
mmol) were added and the reaction mixture was refluxed overnight. The reaction
IS mixture was diluted with ethyl acetate and washed with brine. The organic
layer
was separated and dried over sodium sulfate, filtered, and concentrated under
reduced pressure. The crude material was purified by column chromatography
eluting with 96: 3: 1 methylene chloride: methanol: cone. ammonium hydroxide
to yield 144, (0.300 g, 33%), mp 53-55 °C; HPLC: Inertsil ODS-3V C18,
40:30:30 [KHaP04 (0.01 M, pH 3.2): CH30H: CH3CN], 264 nm, Rt 6.8 min,
92.6% purity; MS (ESI): m/z 449 (M+H, 100), 381 (1.4), 353 (11.8), 226 (13),
225 (33.1), 212 (15), 177 (13.5), 164 (7.8).
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EXAMPLE 47
Synthesis of NZ-((1-ethyl-2 pyrr°olidinylJ-Nø-(3 fluoro-4-
methoxyphenyl)-6-~(S)-
2-(naethoxyrrrethyl)-I pyrr-olidinylJ-1,3,5-tr-iazine-2,4-diamirte (145)
F
OMe
CI HNI
N~N 3-fluoro-p-anisidine, CH3CN, N~N
CI~N~CI iPr2NEt, -20 C; rt 1 h CI~N~CI
124
F
N~ , OMe
1. ~NHz HN
CH3CN, iPr2NEt, rt, I
overnight N~N
1
H ~N~N~N
2. ~",~~OMe N H H...
CH3CN, 1,4-dioxane OMe
iPr~NEt, 50 C, overnight
145
Cyanuric chloride (11.07g, 60 mmol) was dissolved in 40 mL
CH3CN and was cooled to about-20 °C. To this was added DIEA (11.5
mL, 60
mmol) followed by 3-fluoro-4-methoxyaninline (8.47g, 60 mmol) in 20 mL
CH3CN (reaction froze). The reaction was allowed to warm to room temperature
after about 1 hour at -20 °C. TLC (2% CH30H/CH2Cla) and mass
spectroscopy
indicated the presence of the compound 124. The reaction mixture was cooled to
about 0 °C before adding DIEA (11.5 mL, 66 mmol). 2-Aminomethyl-1-
ethylpyrrolidine (7.77 g, 60 mmol) in CH3CN (10 mL) was added. The reaction
was allowed to warm to rt and stirred overnight. Then DIEA (11.5 mL, 66 mmol)
and S-(+)-2-methoxyethylpyrrolidine (6.91 g, 60 mmol) in 20 mL 1,4-dioxane
were added. The reaction was heated at about 50 °C overnight. The
solvent was
removed in vacuo, and the resulting residue was purified by flash
chromatography on silica gel packed in ethyl acetate. The front running
impurities were removed and subsequently the eluent was increased in polarity
to
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10% CH30H: ethyl acetate. The material collected from the column was then
dissolved in water and extracted in CH2C12 (4 times), dried over MgS04, and
concentrated to dryness to give a brown solid 145 (9.7 g, 27.6% yield), 71-72
°C;
HPLC: Inertsil ODS-3V C18, 40:30:30 [I~HZP04 (O.OlM, pH 3.2): CH30H:
CH3CN], 264 nm, RL 5.37 min, 90.3 % purity;'H NMR (600 MHz, CDCl3, 55
°C)
8 7.69 (s, 1H), 7.08 (d, J= 7.8 Hz, 1H), 6.86 (t, J= 9 Hz, 1H), 4.29 (s, 1H),
3.90
-3.96 (m, 1H), 3.84 (s, 3H), 3.63-3.81 (m, 6H), 3.35 (s, 3H), 3.23-3.25 (m,
1H),
2.85 (broad s, I H), 2.78 (broad s 1 H), 2.14 (broad s, 2H), 1.89-2.04 (m,
6H), 1.37
(apparent t, J= 7.2 Hz, 3H);'3C NMR (150.8 MHz, CDC13, 55°C) S 165.8,
163.8
(2C), 152.3 (d, J~_f= 243.5 Hz), 143.0 (142.9, rotamer or diastereumer), 133.7
(133.67, rotamer or diastereomer), 115.0, 114.4, 109.1 (108.9, rotamer or
diastereomer), 72.8, 66.6, 59.0, 57.0, 56.6, 53.7, 51.0, 46.8, 42.2, 28.4
(28.2,
rotamer or diastereomer), 23.1 (23.0, rotamer or diastereomer), 10.9; MS (ESI)
m/z 460.2 (M+H, 44.7), 251.1 (47.7), 235.1 (27.5), 231.1 (37.4), 230.6 (100),
214.6 (36.5).
EXAMPLE 48
Synthesis of (3-Chloro-4-methoxy~henyl)-(4,6-dichloro-~1,3,SJt~iazin-2 y1)-
amine (101)
OCH3
CI
CI
CI HaN ~ / OCH3 HN
~-. N
N'' ~>---CI N~ ~>---CI
>=N NaOH (aq), ~N
CI acetone, 0-5 C, CI
101
To cyanuric chloride (36.911 g, 200.0 mmol) dissolved in acetone
(250 mL) stirring at approximately 0 - 5° C (ice-water bath), was added
a
solution of 3-chloro p-anisidine (31.528 g, 200.0 mmol) in acetone (150 mL)
followed by the addition of NaOH solution (80 mL, 2.5 N, 200.0 mmol). The
reaction mixture was allowed to stir at approximately 0 - 5° C (ice-
water bath)
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for about 1 hour. The reaction mixture was then poured over crushed ice and
neutralized with 10% HCl (aq). The resulting solid was washed with water and
dried overnight under vacuum to afford 101 (58.3 g, 96%), mp 165 ~C; HPLC:
YMC Pack Pro C18, 40:30:30 [KHZPO~ (0.01M, pH 3.2): CH30H: CH3CN], 264
nm, Rt 24.3 min, 97.8% purity); MS (ESI): J~~lz 305 (M+H, 100), 283 (26.3),
271
(26.9), 269 (75.2), 139 (16.2).
EXAMPLE 49
Synthesis of 6-Chloro-N (3-chloro-4-nZethoxy phenyl)-N'-cycloheptyl-
~l , 3, SJtriazine-2, 4-diamine (133)
OCH3 OCH3
CI ~I \
\
/ /
HN~N HZN~ H ~ \
N
N ~ CI N~ ~-CI
N NaOH (aq), Ha0 N
acetone, reflux, HN
101 3 hr, N2 atm 133
To a sample of compound 101 (20.02 g, 65.6 mmol) in acetone
(200 mL) was added cycloheptylamine (8.3 mL, 65.5 mmol) in acetone (55 mL)
slowly by addition funnel at rt. Then water (66 mL) was added followed by
aqueous sodium hydroxide (26.2 mL, 2. 5 N, 65.5 mmol) by addition funnel. The
reaction mixture was heated at reflux under a nitrogen atmosphere for
approximately about 3 hours. The reaction was cooled, diluted with ethyl
acetate,
washed 1 time with water, and finally 1 time with brine. The organic layer was
separated and dried over potassium carbonate/sodium sulfate. The organic layer
was filtered and concentrated in vacuo. The product (24.13 g) was purified by
flash column chromatography (silica gel, 1:4 ethyl acetate: hexanes). The
fractions were combined and concentrated in vacuo to afford 133 as a pale
yellow
solid (17.66 g, 70.5%), mp 146 ~C; HPLC: Inertsil ODS-3V C18, 40:10:50
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[KHZP04 (0.01M, pH 3.2): CH30H: CH3CN], 264 nm, Rt 58.8 min, 99.9%
purity); MS (ESI): rnlz 382 (M+H, 100), 241 (2.8), 226 (8.4), 139 (43.5), 116
(6).
EXAMPLE 50
Syrzthesis of N (3-Chloro-4-naethoxy phenyl)-N'-cycloheptyl-N"-methyl-N"-(1-
methyl piper~idin-4 yl)-(1,3,SJtriazine-2,4,6-tr~iamine (137)
OCH3 OCH3
CI CI CI OMe CI OMe
/ \ / \
HN H~N--CN-CN, HN HN HN
N\-N CI N OH (aq). Ha0 N~N N.CH3 + N~ ~NCHa + N~N OH
HN/~ 1,4-dioxane HN~ ~ HN ~ HN
reflux, 2.5 hr, NZ atm N N
H H
133 137 146 147
To 133 (10.014 g, 26.2 mmol) in 1,4-dioxane (80 mL) was added
slowly methyl-(1-methyl-piperidin-4-yl)-amine (3.8 mL, 26.2 mmol) dissolved in
1,4-dioxane (IS mL) by addition funnel. Then aqueous sodium hydroxide (10.5
mL, 2.5 N, 26.2 mmol) was added by addition funnel followed by water (26 mL).
The reaction mixture was heated at reflux for about 2.5 hours under a nitrogen
atmosphere. The reaction was cooled and diluted with methylene chloride. The
reaction mixture was filtered using vacuum and the white solid 147 removed.
The
filtrate was then washed 1 time with brine. The aqueous layer was back
extracted
1 time with methylene chloride. The organic layers were combined and dried
over potassium carbonate. The organic solution was filtered and concentrated
in
vacuo to afford the crude product (5.89 g). The crude reaction product was
purified by flash column chromatography (silica gel) eluting with 96:3:1
methylene chloride: methanol: 15 M ammonium hydroxide. The fractions were
combined, dried over sodium sulfate/potassium carbonate, filtered, and
concentrated in vacuo to afford 137 as a white solid (3.84 g, 30.9%), mp 104-
105
~C; HPLC: YMC Pack Pro C18, 40:30:30 [KHZP04 (0.01M, pH 3.2): CH30H:
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CH3CN], 264 nm, Rt 13.8 min, 97% purity); MS (ESI): m/z 474 (M+H, 41), 408
(2.3), 364 (2.8), 258 (13), 239 (14), 239 (47.5), 238 (100), 127 (5.3).
EXAMPLE 51
Synthesis of NZ-(3-chloro-4-methoxy phenyl)-Nø-cycloheptyl-1V6-rnethyl-IV6-
piperidin-4 yl-1,3,5-triazirre-2,4,6-tr~iamine (146)
Compound 146 was isolated as a by-product via column
chromatography (silica gel, 96:3:1 methylene chloride: methanol: conc.
ammonium hydroxide, mp 114-116 °C; TLC (silica gel, 90: 9: l, CH2Cl2:
CH30H; conc. NH40H), Rf 137 0.31 and Rf 146 0.15; HPLC: Inertsil ODS-3V
C 18, 40:30:30 [KHzP04 (0.01 M, pH 3.2) :CH3OH: CH3CN], 264 nm, R~ 10.7
min, 91.1% purity); MS (ESI): rrrlz 460 (M+H, 25.4), 364 (17.9), 292 (2), 273
(17.1), 272 (37.9), 252 (44), 251 (100), 231 (2.2), 157 (10.54), 118 (2.8).
EXAMPLE 52
Synthesis of 4-(3-Chloro-4-methoxy phenylamino)-6-cycloheptylamino-1,3,5-
tr~iazin-2-of (147)
Compound 147 was isolated as a by-product by vacuum filtration
prior to isolation of 137, white solid, mp >310 °C; MS (ESI); m/z 727
([2(363)+H], 1.2, 364 (M+H, 100).
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EXAMPLE 53
Synthesis of N (I-Aza-bicyclo(2.2.2Joct-3 yl)-N'-(3-chloro-4-methoxy phenyl)-
N")1-ethyl pyrrolidin-2 ylmethyl)-~1,3,SJtriazine-2,4,6-triamine (148)
c1
CI H~N OMe
OMe 1
~N ~
~~// \
HN
HN DIEA, CH3CN, 1,4-dioxane, rt,
overnight, NZ atm N~N
N N
HN N NH
CI N CI ~NHZ I
2. N N 'N
101 DIEA, CH3CN, reflux, overnight,
- Nz atm 148
To 101 (3.056 g, 10.0 mmol) dissolved in anhydrous acetonitrile
(30 mL) at about 0 °C was added a solution of 2-(aminomethyl)-1-
ethylpyrrolidine (1.5 mL, 10.0 mmol) in anhydrous acetonitrile (5 mL) followed
by addition of a D1EA (1.9 mL, 11.0 mmol). The reaction mixture was allowed
to warm to room temperature and was stirred at room temperature overnight
under nitrogen. Then DIEA (1.9 mL, 11 mmol) was added which was followed
by addition of 3-aminoquinuclidine dihydrochloride (1.962 g, 10.0 mmol) in 1,4-
dioxane (5 mL). The reaction mixture was allowed to stir at reflux overnight
under nitrogen. The reaction mixture was extracted 2 times with
dichloromethane and 1 time with ethyl acetate. The combined organic layers
were washed one time with brine and dried over anhydrous potassium carbonate.
The organic layer was with 20% HCl (aq). The. aqueous layer was neutralized
with 2.5 N NaOH (aq) and then extracted 3 times with ethyl acetate. The
combined organic layers were washed 1 time with brine, dried over potassium
carbonate, concentrated on a rotary evaporator and allowed to dry overnight
under vacuum. Column chromatography (silica gel, 85:14:1 dichloromethane:
methanol: conc. ammonium hydroxide) yielded a pale white solid 148 (100 mg,
2%), mp 83 °C; HPLC: Inertsil ODS-3V C18, 40:30:30 [I~H2P04 (0.01M, pH
3.2) :CH30H: CH3CN], 264 nm, Rt 8.1 min, 71.2% purity); MS (ESI): m/z 488
(M+H, I 8.7), 280 (100), 245 ([M+2H]++, 37.4), 236 (23.5).
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EXAMPLE 54
Synthesis ofN~-(3-chloro-4-diethylamino phenyl)-lV~-cycloheptyl-lV~-(I-ethyl
pyrrolidin-2 ylmethyl)-I, 3, S-tf-iazine-2, 4, 6-triamine (149)
ci
HzN ~ ~ N(CHZCH3)2. HCI
DIEA, CH3CN, -20~C then rt, NZ atm
CI~N 2. ~NHZ
N ~>-CI
>=N DIEA, CH3CN, rt, overnight, NZ atm
CI
~NHz
DIEA, CH3CN, reflux, NZ atm
149
To a mixture of cyanuric chloride (1.8 g, 9.7 mmol) in CH3CN at
about -20°C was added 2-chloro N,N diethyl phenylene-1,4-diamine
hydrochloride (2.35 g, 10 mmol) in CH3CN followed by the addition of N,N
diisopropylethylamine (DIEA) (1.75 mL, 10 mmol) and stirred for an hour. The
reaction mixture was then allowed to reach room temperature for about 1 hour.
Then cycloheptylamine (1.25 mL, 9.8 mmol) and DIEA (1.75 mL, 10 mmol)
were added and the reaction mixture was stirred overnight at rt. Then, 2-
(aminomethyl)-I-ethylpyrrolidine (1.45 mL, 10 mmol) and DIEA (1.75 mL, 10
mmol) were added and the reaction mixture was refluxed overnight. The reaction
mixture was diluted with ethyl acetate and washed with brine. The organic
layer
was separated and dried over sodium sulfate, filtered, and concentrated under
reduced pressure. The crude material was purified by column chromatography
(silica gel) eluting with 96: 3: 1 methylene chloride: methanol: conc.
ammonium
hydroxide to yield 149 (0.800 g, I S%) as a white solid, mp 84-85°C;
HPLC:
Inertsil ODS-3V C18, 40:30:30 [I~H2P04 (0.01 M, pH 3.2): CH30H: CH3CN],
264 nm, Rt 9.5 min, 96% purity; MS (ESI): m/z 515 (1VI+H, 9.4), 259 (16.8),
258
(55.1), 257 (100).
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EXAMPLE 55
Syr~tl7esis ofN2-cycloheptyl-N~-(2-dirnethylarnino-ethyl)-1V6-(3 fluoro-4-
methoxy-
~henyl)-l, 3, 5-tr~iazirre-2, 4, 6-triamine (1 SO)
CI 1. a) 3-fluoro-p-anisidine, CH3CN, -10 C
N~N b) iPr2NEt, 45 min then rt, 45 min
~I
CI~N~CI 2. cycloheptylamine, iPr~NEt, RT, overnight
3. N,N-dimethylethylenediamine, iPr2NEt,
reflux, overnight
150
Cyanuric chloride (1.84 g, 10 mmol) in CH3CN (20 mL) was
cooled to about -10 °C was added 3-fluoro p-anisidine (1.41 g, 10 mmol)
followed by DIEA (1.8 mL, 10 mmol). The reaction was stirred for about 45 min
then at room temperature for about 45 min under an N2 atmosphere.
Cycloheptylamine (1.26 mL, 10 mmol) was added followed by DIEA (1.8 mL,
10 mmol) and the reaction was stirred at room temperature overnight. N, N
dimethylethylenediamine (1.1 mL, 10 mmol) was added followed by DIEA (1.8
mL, 10 mmol) and the mixture was heated at reflux under N2 overnight. The
reaction was diluted with ethyl acetate, washed with brine, and dried over
anhydrous K2C03. The material (1.178 g) was purified by silica gel column
chromatography to afford a solid 150 (1.178 g, 28%), mp 73-76 °C; HPLC:
lnertsil ODS-3V C18, 40:30:30 [KH2PO4 (0.01M, pH 3.2): CH30H: CH3CN],
264 nm, RL 10.8 min, 95.1% purity; MS (ESI): m/z 418 (M+H, 100), 373 (11.9),
322 (7.8), 277 (6.8), 162 (3.6).
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EXAMPLE 56
Syntlaesis of (j4-cycloheptylarnir~o-6-jl-ethyl pyrrolidin-2 yhrtethyl)-
arninoJ-
1, 3, 5-triazin-2 yl) phenyl-amino)-acetonitrile (1 SI)
CI 1. N-phenyl glycinonitrile, CH3CN, DIEA,
N~N -20 C, 1 h; rt, 1 h
CI~N~CI 2. cycloheptylamine, DIEAt, rt, overnight
3. 2-aminomethyl-N-ethylpyrrolidine, DIEA,
reflux, overnight
NC~N
N~N
I
HN~N~NH
151
To cyanuric chloride (1.84 g, 10 mmol) in CH3CN (20 mL) at
about -10 to -20°C was added DIEA (1.75 mL, 10 mmol) and N phenyl
glycinonitrile (1.3 g, 10 mmol), and stirred for about 1 hour. The reaction
mixture was then allowed to reach room temperature for an hour. To this
reaction
mixture, DIEA (1.75 mL, 10 mmol) and cycloheptylamine (1.25 mL, 10 mmol)
were added and the reaction mixture was stirred overnight at rt. Then, DIEA
(1.75 mL, 10 mmol) and 2-aminomethyl-N ethylpyrrolidine (1.45 mL, 10 mmol)
were added and the reaction mixture was refluxed overnight. The reaction
mixture was worked-up, isolated, and then purified by column chromatography
(silica gel) eluting with 96: 3: 1 methylene chloride: methanol: cone.
ammonium
hydroxide to yield 151, (3 g, 66%), mp 52-54 °C; MS (ESI): m/z 449
(M+H,
100), 225 [(M+2H)a+, 22.3).
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EXAMPLE 57
Synthesis of N ~zepan-1 yl-6-chloro-N'-(3-ehloro-4-methoxy phenyl)-
~1,3,SJtriazirre-2,4-diantine (152)
OCH3
OCH3 CI
CI ~
NHz HN
N ~N
H i-
// ~
~N ~--CI
N
N~ ~~-CI N N
~N H
CI NaOH (aq),
Acetone,
reflux
3 hrs, N~
atm
101 152 153
To 101 (6.03 g, 20.0 mmol) dissolved in acetone (75 mL) was
added a solution of 1-aminohomopiperidine (2.3 mL, 20.0 mmol) in acetone (10
mL) followed by addition of NaOH (8.0 mL 2.5 N NaOH solution, 20.0 mmol)
and 20 mL of water. The reaction mixture was allowed to stir at reflux
overnight
under nitrogen. The reaction mixture was extracted 3 times with
dichloromethane; the combined organic layers were washed with brine and dried
over potassium carbonate. The sample was concentrated on a rotary evaporator
and the resulting oil was dried overnight under vacuum. Column chromatography
(96:3:1 dichloromethane: methanol: conc. ammonium hydroxide) yielded a light
purple solid 152 (1.2 g, 16°J°), mp 139 °C; TLC (silica
gel, 96: 3: 1, CH2C12,
CH30H, conc. NH40H), Rf 0.31; HPLC: Inertsil ODS-3V C18, 40:30:30
[I~H2P04 (0.01 M, pH 3.2): CH30H: CH3CN], 264 nm, Rt 52.5 min, 94.9%
purity; MS (ESI): m/z 383 (M+H, 100).
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EXAMPLE 58
Synthesis of N"-(3-chloro-4-methoxy phenyl)-N,N'-bis perhydro-azepin-I y1-
l, 3, 5-t~°iazine-2, 4, 6-triamine (153)
Compound 153 was isolated as a by-product (2.3 g) by column
chromatography (silica gel, 96: 3: 1, CH2C12, CH30H, conc. NH40H), mp 199
°C; TLC (silica gel, 96: 3: l, CHZC12, CH30H, cone. NH40H), Rf 0.1 l;
HPLC:
Inertsil ODS 3V C18, 40:30:30 [KHZP04 (O.OlM, pH 3.2) :CH30H: CH3CN],
264 nm, Rt 15 min, 86% purity); MS (ESI): m/z 461 (M+H, 100), 366 (19.7), 365
(19.6), 232 (l l), 231 (27.3).
EXAMPLE 59
Synthesis of NAzepan-1 yl-N'-(3-chloro-4-methoxy phenyl)-N"-(1-methyl-
piperidin-4 yl)-(1,3,SJtriazine-2,4,6-triamine (154)
OCH3 OCH3 OCH3
CI ~ CI ~ CI
HN~N Cti,HN--( ;N-CH, HN~N CH3 HN~N CH
~ -I- ~
~ N 3
~
N N N
~-CI ~-N >--
~N NaOH (aq), acetone, ~=N ~=N
HN reflux overnight, NZ atm HN
HN ~
N N N N N
U U ~H3 U
152 154 155
To 152 (0.2007 g, 0.5 mmol) dissolved in THF (10 mL) was added
a solution ofN methyl-4(methylamino)piperidine (0.07 mL, 0.5 mmol) in THF (1
mL) followed by the addition of DIEA (1.0 mL, 0.55 mmol) in acetonitrile (1
mL). The reaction mixture was allowed to stir at reflux overnight under
nitrogen.
The reaction mixture was extracted 3 times with dichloromethane; the combined
organic layers were washed with brine and dried over potassium carbonate. The
sample was concentrated on a rotary evaporator and the resulting oil was dried
overnight under vacuum. Column chromatography (90:9:1 dichloromethane:
methanol: cone. ammonium hydroxide) yielded a light yellow solid 154 (65 mg,
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27%), mp 100 °C; TLC (silica gel, 90: 9: 1 CH2CIa: CH30H, conc. NH40H),
Rf
0.36; MS (ESI): m/z 475 (M+H, 23.2), 378 (11.6), 258 (68.9), 239 (52.2), 238
(l 00).
EXAMPLE 60
Synthesis ofNd-(3-claloro-4-rnethoxy phenyl)-IV6-methyl-Na perhydro-azepin-1
yl-
IV6 piperidin-4 y1-1, 3, 5-triazine-2, 4, 6-triamine (1 SS)
Compound 155 was obtained as a by product (50 mg) of the
reaction via column chromatography (silica gel, 90:9:1 dichloromethane:
methanol: cone. ammonium hydroxide), mp 81 °C; TLC (silica gel, 90: 9:
1
CHZC1~: CH3OH, conc. NH40H), Rf 0.25; MS (ESI): m/z 461 (M+H, 20.3), 430
(2.8), 273 (11.8), 272 (25.5), 251 (100), 236 (4.6), 215 (4.7).
EXAMPLE 61
Synthesis of N,N'-di-n propyl-N"-(3 fluoro-4-rnethoxy phenyl)-1,3,5-triazine-
2, 4, 6-triarnine (156)
OCH3
F F
1. HZN ~ ~ OMe
CI
>--N DIEA, CH3CN, HN
-20 C; rt
N~ ~~CI ~-N
~N 2. CH3CHZCHZNH~, N
CI DIEA, CH3CN, rt, ~=N
overnight
156
To cyanuric chloride (0.368 g, 2 mmol) in CH3CN at about -20°C
was added 3-fluoro p-anisidine (0.28 g, 2 mmol) in CH3CN followed by the
addition of DIEA (0.39 mL, 2.2 mmol) and stirred for about 1 hour. The
reaction
mixture was then stirred at room temperature for about 1 hour. Then n-
propylamine (1.64 mL, 19.9 mmol) and DIEA (0.39 mL, 2.2 mmol) were added
and the reaction mixture was stirred overnight at rt. The reaction mixture was
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worked up as usual, diluted with ethyl acetate and washed with brine. The
organic layer was separated and dried over sodium sulfate, filtered,
concentrated
under reduced pressure, and compound 156 was purified by silica gel column
chromatography.
mp 53-55 °C; HPLC: Inertsil ODS-3V C18, 40:30:30 [KHaP04 (0.01 M, pH
3.2)
:CH30H: CH3CN], 264 nm, Rt 12.6 min, 93.7% purity; MS (ESI): m/z 335
(M+H, 100), 331 (1.5), 126 (I).
EXAMPLE 62
S'y~thesis ofN,N'-a'icyclop~~opyl-N"-(3 fheoro-4-methoxy phetzyl)-1,3,5-
triazine-
2,4,6-t~~iamine (157)
OCH3
F F
1. H2N ~ / OMe
CI
>-N DIEA, CH3CN, -20 C; rt HN
N~ ~>-CI ~-N
>=N 2. cyclopropylamine, DIEA, N
CI CH3CN, rt, overnight ~N
D--N
H
157
To cyanuric chloride (0.368 g, 2 mmol) in CH3CN at about -20°C
was added 3-fluoro p-anisidine (0.28 g, 2 mmol) in CH3CN followed by the
addition of DIEA (0.39 mL, 2.2 mrnol) and stirred for about 1 hour. The
reaction
mixture was then stirred at room temperature for about 1 hour. Then
cyclopropylamine (1.39 mL, 20 mmol) and DIEA (0.39 mL, 2.2 mmol) were
added and the reaction mixture was stirred overnight at rt. The reaction
mixture
was worked up as usual, diluted with ethyl acetate and washed with brine. The
organic layer was separated and dried over sodium sulfate, filtered,
concentrated
under reduced pressure, and compound 157 was purified by silica gel column
chromatography (200 mg, 30%), mp 91-92 °C; HPLC: Inertsil ODS-3V C18,
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40:30:30 [KHaP04 (0.01 M, pH 3.2): CH30H: CH3CN], 264 nm, Rt 8.6 min,
99.1 % purity; MS (ESI): m/z 331 (M+H, 100), 305 (0.8), 151 (.3).
EXAMPLE 63
Synthesis of N2-Cycloheptyl-N°-(3 fluoro-4-methoxy phehyl)-lV~-methyl
lV6-(1-
naethyl piperidin-4 y1)-1, 3, 5-triazine-2, 4, 6-triamine (158)
F
HZN ~ ~ oMe OMe OMe
F F
DIEA, CH3CN, 1,4-dioxane -10°C
then rt, NZ atm ( , I i
(/~~y HN HN
CN~ ~CI 2~ V NH= N~- ~N.CH3 + N~ ~N.CH3
~N DIEA, CHaCN, rt, overnight, N2 atm HN N ~ HN~N
CI
3. H3C-N--( N-CH3 ~ CH H
H ~/
DIEA, CH3CN, reflux, NZ atm
158 159
To cyanuric chloride (0.180 g, 1 mmol) in 1,4-dioxane (1 mL) at
about -10 to -20°C was added N,N diisopropylethylamine (DIEA) (0.19 mL,
l mmol) in CH3CN (1 mL) and 3-fluoro p-anisidine (0.14 g, 1 mmol) in CH3CN
(1 mL) and stirred for about I hour. The reaction mixture was then stirred at
room temperature for about I hour. Then a solution of cycloheptylamine (0.13
mL, 1 mmol) and DIEA (0.19 mL, 1 mmol) in CH3CN (0.5 mL) was added and
the reaction mixture was stirred overnight at rt. Then, N methyl-
4(methylamino)piperidine (0.15 mL, I mmol) and DIEA (0.19 mL, 1 mmol) in
CH3CN (0.5 mL) were added and the reaction mixture was refluxed overnight.
The reaction mixture was worked-up using saturated sodium bicarbonate, and
brine. The organic layer was separated and dried over sodium sulfate,
filtered,
and concentrated under reduced pressure. The crude material was purified by
column chromatography (silica gel, 90:9:1 dichloromethane: methanol: conc.
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ammonium hydroxide) to give 158 (0.130 g, 28%); TLC (silica gel, 90: 9: 1,
CHZCIZ, CH30H, cone. NH40H), Rf 0.26); ~H NMR (600 MHz, CDCl3, 55
°C) 8
7.74 (br s, 1H), 6.94 (br s, IH), 6.81-6.84 (m, 2H), 4.83 (br resonance, 1H),
4.55
(s, 1H), 3.98 (s, 1H), 3.82 (s, 3H), 2.97 (s, 3H), 2.94 (br d, J= 11.9 Hz,
2H), 2.29
(s, 3H), 2.06-2.10 (m, 2H), 1.93-1.97 (m, 2H), I.84-1.90 (m, 2H), 1.44-1.66
(m,
12H).
EXAMPLE 64
Synthesis of NZ-Cycloheptyl-lV~-(3-fluoro-4-rnethoxy phenyl)-lV6-methyl 1V6-
1 0 piperidin-4 y1-1, 3, S-triazine-2, 4, 6-triamine (159)
Compound 159 was isolated as a by-product (55 mg) by column
chromatography (silica gel, 90:9:1 dichloromethane: methanol: cone. ammonium
hydroxide); TLC (silica gel, 90: 9: l, CHZC12, CH3OH, cone. NH40H), Rf 0.1);
HPLC: Inertsil ODS-3V C18, 40:30:30 [I~H2POø (0.01 M, pH 3.2): CH30H:
CH3CN], 264 nm, Rt 8.3 min, 93.5% purity; MS (ESI): m/z 443 (M+H, 100).
EXAMPLE 65
Synthesis ofN~-cycloheptyl-IVY-(3 fluoro-4-naethoxyphe~cyl)-IV6-methyl lV6-(1-
methyl piperidin-4 yl)-1,3,5-triazine-2,4,6-triamir~e, hydrogen chloride salt
(160)
F OMe HCI
HN
N~N NCH3 HCI, CH30H
N ~ rt, 10 min
HN
N
CH3
171 160
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To 171 in dry methanol (1 mL, prepared according to parallel
synthesis Method C using the appropriate monomers, as disclosed herein) was
added HCl (0.3 mL, 0.3 mmol, 1 M in diethyl ether) by syringe under a N2
atmosphere. The mixture was stirred for 10 min at room temperature,
concentrated and dried in vacuo overnight to give an off white solid 160
(0.131
g) that is water soluble, mp 189-190 °C (at 160 °C sample turns
brown); HPLC:
Inertsil ODS-3V C18, 40:30:30 [KHZPO4 (0.01 M, pH 3.2): CH30H: CH3CN],
264 nm, Rt 7.3 min, 89.1 % purity.
EXAMPLE 66
Synthesis of(N (3-Chloro-4-methoxy phe>zyl)-N'-eycloheptyl N"-methyl N"-(1-
naethyl piperidir~-4 yl)-(1,3,SJtrizaine-2,4,6-triarrZine (161)
HCI
OCH3
CI
to
1 M HCI/Ether
HN
N~ ~-NH3 CH30H
N
HN
CH3
137 161
To 137 (0.473 g, 1.0 mmol) dissolved in methanol (5 mL) was
added 1.0 M hydrochloric acid in diethyl ether (1.0 mL, lmmol). The reaction
mixture was allowed to stir for about 1 hour at room temperature. The reaction
mixture was then concentrated on a rotary evaporator. The resulting solid was
dissolved in water, filtered and concentrated on the rotary evaporator. The
sample was freeze dried under vacuum and a solid 161 (359.1 mg, 70%) was
collected, mp 173-176 °C.
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EXAMPLE 67
Synthesis of NZ-(3-chloro-4-diethylarrrirzo phenyl)-lVd-cycloheptyl-N6-(1-
ethyl-
pyrrolidin-2 ylrnetlZyl)-I, 3, 5-triazirre-2, 4, 6-triamir~e hydrogen chloride
salt (163)
HG
CI ' N~
HCI, diethyl ether
CH30H, rt
162 163
To 162 (1.0 g, 2 mmol, prepared according to parallel synthesis
method A with the appropriate monomers, as disclosed herein) in methanol (10
mL) was added HCl (2.5 mL, 2.5 mmol, 1 M) in diethyl ether and stirred. The
reaction mixture was evaporated. It was then dissolved in water, filtered,
evaporated in vacuo, and dried over night under vacuum to afford a solid 163
(1.l
g, 93%).
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EXAMPLE 68
Synthesis of NZ-cycloheptyl-N~-(1-ethyl pyrrolidin-2 ylmethyl)-N6-(3 fluoro-4-
r~~ethoxypherryl)-I, 3, 5-triazir~e-~, 4, 6-triarrzirre hydrogen chloride salt
(164)
F OMe H01
HCI, ether, methanol ' HN
rt, 1 h Nz N~ ~>--N
N
HN
-J
130 164
To 130 (2.285g, 5 mmol) in dry methanol (10 mL) was added HCl
(5 mL, 5 mmol, 1 M in diethyl ether) and stirred at room temperature for about
1
hour. The reaction was evaporated in vacuo, dissolved in water, filtered,
evaporated and then dried under vacuum overnight to afford a solid 164 (2.396
g,
97%), mp 131-133 °C; HPLC: Inertsil ODS-3V C18, 40:30:30 [KHaP04 (0.01
M,
pH 3.2): CH30H: CH3CN], 264 nm, Rt 7.9 min, 98.2% purity.
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EXAMPLE 69
Synthesis of N~-(cyclohexylrrrethyl)-N4-~(1-ethyl-2 pyr~rolidinyl)methylJ 1V6-
(4-
fluor~o-3-rnethoxyphenyl.)-1,3,5-triazine-2,4,6-triamine hydrogen chloride
salt
(165)
F OMe F OMe HCI
HN HCI, diethyl etherHN
N N rt, 1 h, NZ N N
N
HN ~ N N
b H
b
126 165
To 136 (0.457 g, 1 mmol) in dry diethyl ether was added HC1 (1
mL, 1 mmol, 1 M in diethyl ether). A precipitate formed immediately. The
mixture was stirred at room temperature for about 1 hour, and then
concentrated
in vacuo. The resulting material was dissolved in water, filtered, evaporated,
and
dried overnight in vacuo to give a solid 165 (0.400 g,.81%), mp 85 °C;
HPLC:
Inertsil ODS-3V C18, 40:30:30 [I~HZP04 (0.01 M, pH 3.2): CH30H: CH3CN],
264 nm, Rt 8.2 min, 89.6% purity;
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EXAMPLE 70
Synthesis of (~4-cycloheptylamirro-6-((1-ethyl pyrrolidin-2 ylmethyl)-aminoJ-
1,3,5-triazi~-2 y1) phenyl-amino)-acetonitrile hydrogen chloride salt (166)
Hci
Nc~ / \
HCI, diethyl ether NC~N
N
N N N rt, 1 h N~ ~~--N
N
HN N ~ ~ HN
151 166
To 151 (0.448 g, I mmol) in dry diethyl ether (2 mL) was added
HCI (1 mL, I mmol, 1 M in diethyl ether). The mixture was stirred at room
temperature for about 1 hour, and then concentrated in vacuo. The resulting
material was dissolved in water (S-10 mL), filtered, evaporated, and dried
overnight under vacuum to give a solid 166 (0.418 g, 86°fo), mp 125-127
°C;
HPLC: Inertsil ODS-3V C18, 40:30:30 [I~HH~P04 (0.01 M, pH 3.2): CH30H:
CH3CN], 264 nm, Rt 6.9 min, 73.4°J° purity.
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EXAMPLE 71
Synthesis of 1V~-cycloheptyl-1Vø-(3 fluoro-4-methoxy phenyl)-1V6-methyl lV~-(1-
methyl piperidirt-4 yl)-1,3,5-triazitze-2,4,6-triamine r»aleate salt (167)
OCH OCH3 maleate
3
HOzC~CO2H
HN~ HN~
N// ~NCH3 CH30H N// ~NCH3
N N
HN ~ HN
N
CH3 CH3
158 167
Compounds 158 (100.3 mg, 0.219 mmol) and malefic acid (25.4
mg, 0.219 mmol) were dissolved in CH30H (2 mL) and stirred at room
temperature under a N~ atmosphere for about 75 min. The reaction mixture was
filtered through a cotton plug and concentrated in vacuo to afford a solid
167,
0.1239 g, mp 99-100 °C. In a qualitative test, this material was water-
soluble.
HPLC: Inertsil ODS-3V C18, 40:30:30 [KH2P04 (0.01M, pH 3.2): CH30H:
CH3CN], 264 nm, R~ 7.7 thin, 87.9 % purity.
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EXAMPLE 72
Synthesis of Na-cycloheptyl-N~-(3 fluoro-4-methoxy phenyl)-lV6-methyl-IV6-(I-
methyl pipet~idin-4 y1)-1, 3, 5-triazine-~, 4, 6-triamihe citYate salt (168)
c~cH3 citrate
F
I a
HOC(COZH)(CHZCOaH)a HN~
s N// / NCH3
CH OH, RT
N
H
N
~CH3
158 168
Compounds 158 (100 mg, 0.219 mmol) and citric acid (42.1 mg,
0.219 mmol) were dissolved in CH30H (2 mL) and stirred at room temperature
under a N2 atmosphere for about 2 hours. The reaction mixture was filtered
through a cotton plug and concentrated in vacuo to afford a solid 168 (0.1387
g),
mp 125 °C. In a qualitative test, this material was water insoluble.
HPLC: Inertsil
ODS-3 V C 18, 40:30:30 [KHaP04 (0.01 M, pH 3.2): CH30H: CH3CN~, 264 nm,
Rt 7.7 min, 90.1% purity.
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EXAMPLE 73
Synthesis ofN~-cycloheptyl-IV4-(3 flZCOro-4-methoxy phenyl)-IV6-methyl-lV6-(1-
methyl piper idin-4 y1)-1, 3, 5-triazine-~, 4, 6-triamine succinate salt (169)
OCH3 succinate
F
,COzH
HOzC~/ HN~
CH OH
a N// / NCH3
N
HN
NCH3
169
Compounds 158 (101.5 mg, 0.219 mmol) and succinic acid (24.8
mg, 0.219 mmol) were dissolved in CH30H (2 mL) and stirred at room
temperature under a NZ atmosphere for about 75 min. The reaction mixture was
filtered through a cotton plug and concentrated in vacuo to afford a solid 169
(0.1248 g), mp 81°C. In a qualitative test, this material was water-
soluble.
HPLC: Inertsil ODS-3V C18, 40:30:30 [ICHH~P04 (O.OlM, pH 3.2): CH30H:
CH3CNJ, 264 nm, Rt 7.6 min, 89.8 % purity.
EXAMPLE 74
Synthesis of N (3-Bromo-4-methoxy phenyl) N'-cycloheptyl N"-methyl N"-(1-
methyl piperidi~-4 yl)-~1,3,SJtriazirre-2,4,6-triamine hydrogen chloride salt
(170)
To 123 (1.0 mmol) dissolved in methanol (5 mL) was added 1.0 M
hydrochloric acid in diethyl ether (1.0 mL, lmmol). The reaction mixture was
allowed to stir for about I hour at room temperature. 'The reaction mixture
was
then concentrated on a rotary evaporator. The resulting solid was dissolved in
water, filtered and concentrated on the rotary evaporator. The sample was
freeze
dried under vacuum and a solid 170 (70%) was collected
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EXAMPLE 75
Alternative Synthetic Route to Trir(amino) 1,3,5-T>~iazine Cofnpounds
The following reaction scheme represents a proposed and alternative synthetic
routes to 1,3,5-triazines.
X 1
N~N ~' R12NH, iPrZNEt, CH3CN
N' N
X~N~X 2. R~2NH, iPr2NEt, CH3CN R32N~N~NR2~
3. R3 NH, iPr~NEt, CH3CN
X=Br, CI, F, I, OS02CF3,
OSO~CH3, OS02C6H4p-CH3
OP(O)(OEt)~
This scheme represents a modification of the synthetic route
described in the patent to prepare the tris-amino substituted 1,3,5-triazines.
Alternative leaving groups, X, could be used as compared to cyanurie chloride
(X=Cl) in the SNAr reaction with a sequential addition of a nucleophilic amine
in
the presence of an acid (proton) scavenger to afford the tris-substituted
1,3,5-
triazine with the desired combination of amino groups.
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EXAMPLE 76
Alternative Synthetic Route to Ts°is(arniuo) 1,3,5-Triazine
Compounds
The following reaction scheme represents a proposed and
alternative synthetic routes to 1,3,5-triazines.
X NR12
1. R~2NH, base, solvent ~
N~N N! 'N
I
X~N~X ~~ R22NH, base, solvent R3~N~N~NR2~
3. R3~NH, base, solvent
X= F, Br, CI
base=acid scavenger=R3N, NaOH, KOH, NaHC03,
K2C03, Na2C03, resin-NR2, NaH, KH, RLi
solvent=compatible with base=CH3CN, THF, 1,4-dioxane,
Et20,DMS0, DMF, CH2CI2, CHCI3, CICH2CH2CI, C6H5CH3, H2O
This scheme represents a modification of the synthetic route
described in the patent text to prepare the tris-amino substituted 1,3,5-
triazines.
Bases, including excess amine reagent RaNH, could be used as acid (proton)
scavengers alternatively to the Hunig's base (iPr2NEt) used routinely in our
procedure. These bases can include other organic tertiary amine bases or
ionic,
inorganic bases. One can use strong bases (NaH, KH, or RLi) to first
deprotonate
the amino monomer before addition to the cyanuric-X substrate. Additionally,
one can use a solid supported base (e.g., resin-NR2, a modified Hiinig's base)
as a
proton scavenger. This potentially enables an easier isolation procedure and
cleaner reaction products. Logically, one would use the appropriate solvent or
combination of solvents that is compatible with the base of choice for this
procedure.
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EXAMPLE 77
Alter°f7ative Synthetic Route to Tris(amino) 1,3,5-Triazine
Compounds
The following reaction scheme represents a proposed and
alternative synthetic routes to 1,3,5-triazines.
1. aldehyde or ketone (R~), borohydride,
NH2 solvent, pH control NR12
N~N N~N
H2N~N~NH2 2. aldehyde or ketone (R2), borohydride, Ra N~N~NR2
solvent, pH control 2 2
3. aldehyde or ketone (R3), borohydride,
solvent, pH control
borohydride=NaCNBH3, NaBH(OAc)3, NaBH4, or BH3
solvent=THF, 1,4-dioxane, CH30H, CH3CH2OH, CH2CI2,
or CICH2CH~CI
This scheme represents a modification of the synthetic route
described in the patent to prepare the tris-amino substituted 1,3,5-triazines.
Using melamine as the starting material, the method outlined would involve
three
sequential reductive amination procedures. With control of addition,
temperature, and pH, the choice of aldehydes or ketone, one can prepare tris-
amino substituted triazines with the desired combination of amino groups.
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EXAMPLE 78
Alternative Synthetic Route to Tris(arnino) 1,3,5-Triazine Compounds
The following reaction scheme represents a proposed and
alternative synthetic routes to 1,3,5-triazines.
Scheme D
X
N~N
NH3 X~N~X
--L-G --L-NH2
X=Br, CI, F, I
L=linker, cleavable
G=leaving group
NR~2
N_ 1. R~ZNH, iPr2NEt, solvent N
--L-NH--C~ ~N w:.~-L-NH---(~
N 2. RZ~NH, ~Pr2NEt, solvent N 2
X NR ~
~ JNR~~ NR~2
L-N cleavage N~ reductive amination N=
HEN--C~ N R3~N--~~ N
N-~ or N-alkylation method N
NR22 NR22
This scheme represents a solid phase synthetic approach to
preparing symmetrically or asymmetrically substituted tris-amino substituted
1,3,5-triazines. The resin should possess a readily cleavable linker group (L)
and
a leaving group (G) for attachment of an amino group. The scheme outlines the
synthesis by initially attaching a simple amino group, NHS, by reacting the
resin
with ammonia. Using standard, SNAr chemistry for substitution of a
perhalogenated 1,3,5-triazine, the triazine can be attached to the aminated
resin.
I S Sequential substitutions of the halogens on the triazine core with
functionalized
amines in the presence of an acid scavenger will produce the desired di-amino
substituted 1,3,5-triazine. Cleavage of the triazine from the resin tether
will
afford the tris-amino substituted triazine product. The free NH2 moiety of the
triazine can be further alkylated or fimctionalized using standard chemistry
such
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reductive amination or N-alkylation to give a completely functionalized tris-
amino substituted 1,3,5-triazine.
EXAMPLE 79
Alternative Synthetic Route to Tris(amino) 1,3,5-Triazine Compounds
The following reaction schemes (Schemes A and B) represent
proposed and alternative synthetic routes to 1,3,5-triazines.
Scheme A
1. R~B(OH)2, Pd catalyst, solvent ~HR~
N N N N
2. R2B(OH)2, Pd catalyst, solvent
H2N N NHz g, R3g(OH)2, Pd catalyst, solvent R3HN N NHR2
Scheme S
H)2 1. R~2NH, Cu catalyst, solvent ~ 12
N~ N N~ N
2. R~~NH, Cu catalyst, solvent
(HO)2B N B(OH)~ g. R32NH, Cu catalyst, solvent R32N N NR22
These schemes represent variations on using the Suzuki coupling
to synthesize tris-amino substituted 1,3,5-triazines. As illustrated in Scheme
A,
one can sequentially react the amino groups of melamine with an alkyl or aryl
boronic acid derivative in the presence of the appropriate palladium catalyst,
additives and solvent to afford the symmetric or asymmetric tris-amino
substituted 1,3,5-triazines similar to previously described examples. In
Scheme
B, a tris-boronic acid 1,3,5-triazine can be prepared from cyanuric chloride
or
bromide. This derivative can then be coupled with an aryl or alkyl amine, as
illustrated in previous amine monomer descriptions, in the presence of the
appropriate metal catalysts (e.g., Cu or Pd catalyst), additives and solvent
to
afford the symmetric or asymmetric tris-amino substituted 1,3,5-triazines.
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EXAMPLE 80
P~°oteoglycan Induction
Smooth muscle cells reach quiescence during serum starvation
resulting in a blockade of DNA synthesis. To demonstrate the role of perlecan
(proteoglycan example) in SMC quiescence, cells were starved by removing
serum from the media. The cells used in this Example and the other examples
herein were human aortic SMC, grown in basal medium supplemented with
growth factors, bFGF and epidermal growth factor (EGF) (Clonetics, San Diego,
CA).
SMC secretion of total PGs (proteoglycans) as well as perlecan
were determined in the presence or absence of one or more compounds of the
present invention. PGs were radiolabeled with (35S)sulfate by incubating the
cells with (35S)sulfate for 2 to 6 hours. Media PGs were collected and
purified
IS by DEAE-cellulose chromatography. Cell-associated PGs were assessed by
extracting cells with 50 mM Tris buffer pH 7.4 containing 4 M urea, 1% Triton
X-100, 0.1 mM EDTA and 1 mM PMSF. Aqueous solutions of (35S)sulfate and
(3H)leucine were from Amersham. Control cells have no added compounds
whereas treated cells have one or more compounds of the present invention
added.
To determine changes in PG levels, DEAE-cellulose
chromatography was performed. A DEAF-cellulose column was equilibrated
with 50 mM Tris buffer pH 7.4 containing 4 M urea, 0.1 M NaCI, 0.1 mM
EDTA, 1 mM PMSF and 1% 3[(3-cholamidopropyl)dimethylammonio]-1-
propanesulfonate (CHAPS). The column was washed with the same buffer and
buffer containing 0.25 M NaCI and PG were eluted with the same buffer
containing 0.5 M NaCI. Fractions containing radioactivity (35S~4) were pooled
and dialyzed against MEM overnight and counted.
To determine the relative proportion of HSPG and chondroitin
sulfate/dermatan sulfate proteoglycan (CS/DS PG), an aliquot of the pooled
fraction was incubated in 50 mM sodium acetate buffer pH 5.2 with 1 unit/ml
each of heparanase and heparitinase or with 0.5 units of chondroitin ABC Iyase
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for 16 h at 37° C. Chondroitan ABC refers to different isomeric types
of
chondroitin, e.g. chondroitin A, chondroitin B, and chondroitin C. The
reaction
mixture was precipitated either with 0.5 volumes of 1% cetyl pyridinium
chloride
or with 3 volumes of ethanol to precipitate undigested glycosaminoglycans.
Radioactivity in the supernatant and pellet was determined.
To determine changes in perlecan protein in response to the
presence of a compound, cells were grown in serum-free or serum-containing
media in the presence of (~H)leucine for 24 h (steady state). Cells were
plated at
low density (8 x 104/well in 48 well plate, 30-40% confluency) and cultured
for
24 h (hour). Wells were then replenished with fresh medium containing no
IS
serum or 10% fetal bovine serum (FBS). Following another 24 h incubation,
cells were labeled with (3H)thymidine for 6 h and radioactivity incorporated
into
the DNA was determined by trichloroacetic acid (TCA) precipitation of the cell
lysate. (3H)thymidine was from NEN. Purified PG (0.5 M eluate) were
immunoprecipitated by incubation with an anti-perlecan antibody (100-fold
diluted) followed by precipitation with Protein A-Sepharose.
Immunoprecipitates
were analyzed by 5% SDS-PAGE. Perlecan (Mr>550 leDa) was identified by
autoradiography. Control cells have no added compounds) whereas treated cells
have one or more compounds of the present invention added.
E~fAMPLE 81
Inhibition of Smooth Muscle Cell Proliferation
Purified perlecan from SMC medium by DEAE-cellulose
chromatography was obtained using methods in Example I, and was tested for its
antiproliferative effects on SMC.
The addition of perlecan to serum-containing medium inhibited
SMC growth by 70%. Sub-confluent SMC (40-50% confluence) were incubated
in serum-free medium or 10% serum-containing medium with or without purified
perlecan for 24 h. DNA synthesis was then determined by incubating cells for
another 5 h in medium containing (3H)thymidine. TCA precipitable (DNA)
thymidine counts were determined and expressed as percentage of DNA
synthesis in cells grown in 10%FBS.
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This assay can be used to show the effect of a compound on
perlecan directly by incubating the compound to perlecan first, then
performing
the assay. Alternatively, the cells can be pretreated with at least one
compound
of the present invention to show indirect effects. Control cells have no added
compounds whereas treated cells have one or more compounds of the present
invention added.
EXAMPLE 82
Triazine Compounds in Smooth Muscle Cell Proliferation Assay
Human aortic smooth muscle cells (Clonetics) were used. Cells
were grown in basal medium containing 5% fetal bovine serum supplemented
with growth factors, basic fibroblast growth factor, epidermal growth factor
and
insulin. To determine the effects triazine compounds ofthe present invention
had
on SMC proliferation, cells were plated at low density (4000 cell per well in
a 96
well plate) and cultured for 24 h. The cells were then serum starved for 24 h
to
induce quiescence. Fresh growth medium containing no compound or 10 pM
compound was then added and further incubated for 24 h. Cell number was
determined by using a cell proliferation assay kit (Celltiter96 AQ"e°us
from
Promega).
The effects of different triazine compounds on smooth muscle cell
proliferation are shown in Figure 53. Many of the triazine compounds inhibited
SMC proliferation by greater than 70%.
EXAMPLE 83
Induction and measurement of endothelial heparanase protein
Experiments were carried out on human microvascular endothelial
cells (HMVEC) grown in 48-well plates (~90% confluency). To induce
heparanase activity, culture media was replaced with 200 p1 Dulbecco's
Modified Eagle's medium (DMEM) complemented with 1% bovine serum
albumin (BSA) and with or without stimulants (5 nglml TGF-alpha, 1 ng/ml IL
1 alpha, 200nglml VEGF or other stimulants, cytokines, or inducers as
required).
The secreted proteins were analyzed by SDSIPAGE and heparanase protein was
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detected by immunoblotting using polyclonal anti-human heparanase antibody.
The changes of heparanase expression determined by densitometric analysis. The
induction and measurement of endothelial heparanase protein reported in the
Tables herein were carried out according to this Example.
EXAMPLE 84
Pr°epar°ation of Biotirrylated HS
Heparan sulfate (HS) was biotinylated using biotin with extended
spacer arms using succinimidyl-6-(biotinamido) hexanoate (NHS-LC-Biotin)
obtained from Pierce. About 0.5 ml HS solution (2 mg/ml in NaHC03, pH 8.5)
was mixed with 0.05 ml of a freshly prepared solution of NHS-LC-Biotin in
dimethyl sulfoxide. The mixture was incubated at room temperature for 1 hour.
Unconjugated biotin was removed by centrifugation (10,000 RPM) through
Microcon-3 f Iter (Millipore) followed by dilution with phosphate buffered
saline
(PBS). This procedure was repeated five times to ensure complete removal of
free biotin. Unwanted aldehydes in the reaction were then quenched by
incubation with one milliliter of Tris-glycine buffer(25 mM-183 mM, pH 8.3) at
room temperature for 20 minutes. The mixture was subjected to three rounds of
microfiltration as described above. Biotinylated HS (S mg/ml in PBS) was
aliquoted and stored at -20°C. To obtain maximum biotinylation, a 25-
fold molar
excess of biotin was used. Using HABA reagent, it was determined that the
ratio
of HS to biotin was 1 :2.
The extent of biotinylation of HS was determined using Avidin-
HABA (Pierce Chemical Co). The HABA assay can be used over a wide range
of pH and salt concentrations. HABA (4-hydroxyazobenzene-2'-carboxylic acid)
is a dye that binds to avidin and can serve as an indicator of unoccupied
binding
sites. Avidin combines stoichiometrically with biotin, making it possible to
use
any physiochemical differences between avidin and the avidin-biotin complex as
the basis of a qualitative and quantitative assay method for either component.
When HABA binds to avidin, there is a large spectral change in
the HABA dye. A new absorption band appears at 500 nm, which is
characteristic of the quinoid form of the dye. The avidin-biotin complex does
not
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bind HABA and because the dissociation constant of the complex is so low, the
dye is stoichiometrically displaced by biotin. Consequently, the HABA assay
can
be the basis of both colorimetric and titrimetric assays. The amount of avidin
can
be calculated directly from the increased absorbance at 500 nm, or the dye may
be used as an indicator in a spectrophotometric titration with biotin.
The absorption band that results from the avidin-HABA complex
decreases proportionately when biotin is added. Since biotin has such a high
affinity for avidin, it displaces the HABA dye. The unknown amount of biotin
can be determined by preparing a standard curve using known amounts of biotin
to displace the HABA which bound to avidin, and plotting against the
absorbance
at 500 mu.
HABA solution was prepared by adding 24.2 mg of HABA
(Pierce) to 9.9 ml H20, and then adding 0.1 ml 1 M NaOH. Avidin-HABA
reagent was prepared by adding 10 mg of avidin and 600 g1 of HABA solution to
19.4 ml of phosphate buffered saline. To 1 ml of Avidin-HABA reagent in a
cuvette, 100 p1 of biotinylated HS was added, and the optical density was
measured at 500 nm in a spectrophotometer. A standard curve was determined
using known amounts of HABA. The decrease in optical density of the HABA
following the addition of biotinylated HS was determined.
EXAMPLE 85
Heparahase Assay
Biotin-labeled HS made as described above was digested with
heparanase, under both control and treated conditions, and the reaction
containing
undegraded and degraded HS was bound to in a biotin-binding plate.
Streptavidin, conjugated with an enzyme, was added to the binding plate.
Quantitation of the color reaction measured the amount of available biotin
binding sites. A decrease in color from a known amount reflects HS digestion
by
heparanase. Control conditions have no added compound of the present
invention, and treated conditions have compounds ofthe present invention
added.
A lyophilized powder of heparanase (heparanase III obtained from
Seikagaleu) containing 0.1 units of enzymatic activity was hydrated in 100 p.1
of
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Reaction Buffer (3.33 mM calcium acetate pH 7.0, containing 0.1 mg/ml BSA).
This solution was then diluted to a working concentration of heparanase
solution
(0.01 micro-units to 1 mini-unit) in Reaction Buffer. Enzyme activity was
defined by the manufacturer of the heparanase (Seikagaku) as follows: one unit
of enzyme activity is defined as amount required to generate 1 micromole of
hexuronic acid per minute. Biotin-HS was diluted to a desired concentration in
Reaction Buffer.
To determine heparanase activity, 10 p.1 of heparanase solution,
with or without at least one of the compounds of the present invention, was
mixed with 200 y1 of the biotin-HS substrate in a 96 well plate. The reaction
was
incubated at 43°C for 1 hour. One hundred microliters of the reaction
mixture
was added to a hydrated biotin-binding plate (Chemicon) and incubated at
37°C
for 30 minutes. The biotin-binding plates were hydrated with 200 p.1 of lx
Assay
Buffer (Chemicon). Wells were washed five times with Ix Assay Buffer and
incubated with 100 E~l of 1:3000 diluted Streptavidin-Enzyme Conjugate
(Chemicon) for 30 minutes at 37°C. The wells were washed five times
with lx
Assay Buffer and incubated for 20 minutes with 100 p.1 of Substrate Solution
(Chemicon). Color development in the wells was assessed by measuring the
optical density at 450 nm in a microplate reader (Labsystems, Muliskan Ascent
model). Differences between the control and the treated conditions indicate
the
heparanase modulating activity of the added compound or compounds.
EXAMPLE 86
AGE-induced Inflammatory Respor7.se Determined by IL-6 ELISA
Human aortic endothelial cells (HAEC, Clonetics) were cultured
according to manufacturer in growth medium (Clonetics): basal medium
containing human epidermal growth factor, hydrocortisone, vascular endothelial
growth factor, heparin binding growth factor-B, long R3-insulin-like growth
factor-1, ascorbic acid, gentamicin/amphotericin and 5% FBS. These cell were
allowed to reach at least 90% confluency before subjected to experimental
treatments. Glycated human serum albumin (G-HSA) was from US Biologicals.
Tumor necrosis factor a was from R&D Systems.
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Endothelial cells were treated with control medium or medium
containing 10 to 100 ng/ml TNF-a or 300 ltg/ml glycated-HAS (treated cells or
treatments) for 24 hrs, in control and compound-added duplicates, containing
10
p.M compound.. All treatments, compound-added and controls were carried out
in serum free media containing 0.2% albumin. Media from all conditions were
collected and used for 1L-6 ELISA.
IL-6 ELISA was carried out using human IL-6 DuoSet ELISA
development Icit as described by manufacturer (R&D Systems). Mouse anti-
human II-6 was used as the capture antibody (2ug/ml) and biotinylated goat
anti-
human IL-6 (200ng/ml) was used as the detection antibody. Culture media were
incubated with capture antibody (in 96 well) for 2 h at room temperature.
Wells
were washed three times with wash buffer (0.05% tween-20 in phosphate
buffered saline (PBS) pH 7.4) followed by incubation with detection antibody
for
2 h at room temperature. Following three washes wells were incubated with
Streptavidin-HRP for 20min. Color development was read at 450nm in a
Microplate reader.
The effects of compounds of the present invention on G-HSA
induced IL-6 are shown in Figure 54. G is G-HSA, and C is control, no
treatment
with compounds or G-HSA. Endothelial cells under basal conditions secreted
about 25 pg/ml of IL-6. Incubation of endothelial cells with G-HSA induced a 3
fold increase in 1L-6 secretion by endothelial cells. Addition of compounds of
the present invention, as indicated by each compound's number, to G-HSA
containing media significantly reduced endothelial secretion of IL-6. These
inhibitory effects varied, the most effective compounds showed an ~0% decrease
in IL-6 secretion. These data show the compounds of the present invention have
anti-inflammatory activity.
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EXAMPLE 87
CytotoxicitylLactate Dehydroger~ase Assay
An appropriate number of cells are plated in four 96-well plates,
one plate for "day 0" and three plates for days 1-3. Cells are treated with at
least
one compound of the present invention in varying concentrations with and
without the apoptosis inducer cisplatin (2p,M) ("+cis" or "-cis"). Unteated
cells
are also assayed with and without cisplatin. After transfection, the plates
are
incubated at 37°C overnight.
An appropriate number of cells are plated in four 96-well plates,
one plate for "day 0" and three plates for days 1-3. Cells are treated with at
least
one compound of tile present invention in varying concentrations. The negative
control cells have normal media conditions, a duplicate set of wells is
treated
with the composition in which the compound is provided, but there is no added
compound and the positive control cells are treated with the apoptosis inducer
cisplatin (2yM). All of the cells are transfected with a vector having a
promoter
that is responsive to apoptosis conditions. When apoptosis occurs, the
promoter
is turned on and the lactic dehydrogenase gene is activated and the enzyme
protein is made and active. Activity is easily detected with a color change.
After
transfection, the plates are incubated at 37°C overnight.
About 8 mls of warmed alpha MEM LDH lysis buffer (2% Triton
X100) and about 8 mls of culture media (1/2 dilution) are combined. Two 96-
well v-bottom plates are prepared, one labeled "lysis" and one labeled
"supernatant." To lyse the cells, about 200 p1 Alpha MEM lysis buffer (diluted
%2) is added to one test plate from which the supernatant has been removed and
added to the plate labeled supernatant. After mixing, about 200 p1 of lysed
cells
are transferred to the lysis plate. Both the lysis and supernatant plates are
centrifuged at about 1600 rpm for about 10 min. After centrifugation, about
100
p,1 of both the supernatant or lysate is transferred to corresponding 96-well
flat-
bottomed plates.
The assay for cytotoxicity uses the Cytotoxicity Detection I~it
(LDH) from Roche Diagnostics Corp. (Indianapolis, IN). Using the directionts
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provided, the dye solution is mixed and added each well of the lysate and
supernatant plate and incubated for up to 20-25 min at 15-25°C in the
dark.
The difference in the amount of lactate dehydrogenase released
from cells in untreated cells when compared to cells treated with cisplatin or
compounds of the present invention having cytoxic activity shows the cytotoxic
activity of the compounds tested.
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