Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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GLYCOGEN SYNTHASE KINASE-3 ~ITORS
FIELD AND BACKGROUND OF THE INVENTION
The present invention relates to novel compounds for inhibiting glycogen
synthase kinase-3 (GSK-3) and their use in regulating biological conditions
mediated
by GSK-3 activity and, more particularly, to the use of these compounds in the
treatment of biological conditions such as type II diabetes, neurodegenerative
disorders and diseases and affective disorders.
Protein kinases, the enzymes that phosphorylate protein substrates, are key
players in the signaling of extracellular events to the cytoplasm and the
nucleus, and
take part in practically any event relating to the life and death of cells,
including
mitosis, differentiation and apoptosis. As such, protein kinases have long
been
favorable drug targets. However, since the activity of protein kinases is
crucial to the
well being of the cell, while their inhibition oftentimes leads to cell death,
their use as
drug targets is limited. Although cell death is a desirable effect for
anticancer drugs,
it is a major drawback for most other therapeutics.
Glycogen synthase kinase-3 (GSI~ 3), a member of the protein kinases family,
is a cytoplasmic proline-directed serine-threonine kinase that is involved in
insulin
signaling and metabolic regulation, as well as in Wnt signaling and the scheme
of cell
fate during embryonic development. Two similar isoforms of the enzyme, termed
GSK-3~ and GSK-3~i, have been identified.
GSK-3 has long been considered as a favorable drug target among the protein
kinase family since unlike other protein kinases, which are typically
activated by
signaling pathways, GSK-3 is normally activated in resting cells, and its
activity is
attenuated by the activation of certain signaling pathways such as those
generated by
the binding of insulin to its cell-surface receptor. Activation of the insulin
receptor
leads to the activation of protein kinase B (PKB, also called Akt), which in
turn
phosphorylates GSK-3, thereby inactivating it. The inhibition of GSK-3
presumably
leads to the activation of glycogen synthesis. The intricate insulin-signaling
pathway
is further complicated by negative-feedback regulation of insulin signaling by
GSK-3
itself, which phosphorylates insulin-receptor substrate-1 on serine residues
(Eldar-
Finkelman et al., 1997).
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Therefore, synthetic GSK-3 inhibitors might mimic the action of certain
hormones and growth factors, such as insulin, which use the GSK-3 pathway. In
certain pathological situations, this scheme might permit the bypassing of a
defective
receptor, or another faulty component of the signaling machinery, such that
the
biological signal will take effect even when some upstream players of the
signaling
cascade are at fault, as in non-insulin-dependent type II diabetes.
The regulation of glycogen catabolism in cells is a critical biological
function
that involves a complex array of signaling elements, including the hormone
insulin.
Through a variety of mediators, insulin exerts its regulatory effect by
increasing the
synthesis of glycogen by glycogen synthase (GS). A key event in insulin action
is the
phosphorylation of insulin receptor substrates (IRS-1, IRS-2) on multiple-
tyrosine
residues, which results in simultaneous activation of several signaling
components,
including PI3 kinase (Myers et al, 1992)). Similarly, the activity of glycogen
synthase
is suppressed by its phosphorylation. There is a marked decrease in glycogen
synthase activity and in glycogen levels in muscle of type II diabetes
patients
(Damsbo et al., 1991; Nikoulina et al., 1997; Shulman et al., 1990).
One of the earliest changes associated with the onset of type II (non-insulin
dependent) diabetes is 'insulin resistance. Insulin resistance is
characterized by
hyperinsulemia and hyperglycemia. Although the precise molecular mechanism
underlying insulin resistance is unknown, defects in downstream components of
the
insulin signaling pathway are considered to be the cause.
Glycogen synthase kinase-3 (GSK-3) is one of the downstream components of
insulin signaling. It was found that high activity of GSK-3 impairs insulin
action in
intact cells, by phosphorylating the insulin receptor substrate-1 (IRS-1)
serine
residues (Eldar-Finkelman et al, 1997), and likewise, that increased GSK-3
activity
expressed in cells results in suppression of glycogen synthase activity (Eldar-
Finkelman et al, 1996). Further studies conducted in this respect uncovered
that
GSK-3 activity is significantly increased in epididymal fat tissue of diabetic
mice
(Eldar-Finkelman et al, 1999). Subsequently, increased GSK-3 activity was
detected
in skeletal muscle of type II diabetes patients (Nickoulina et al, 2000).
Additional
recent studies further established the role of GSK-3 in glycogen metabolism
and
insulin signaling (for review see, Eldar-Finkelman, 2002; Grimes and Jope,
2001;
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Woodgett, 2001), thereby suggesting that the inhibition of GSK-3 activity may
represent a way to increase insulin activity in vivo.
GSK-3 is also considered to be an important player in the pathogenesis of
Alzheimer's disease. GSK-3 was identified as one of the kinases that
phosphorylate
tau, a microtubule-associated protein, which is responsible for the formation
of paired
helical filaments (PHF), an early characteristic of Alzheimer's disease.
Apparently,
abnormal hyperphosphorylation of tau is the cause for destabilization of
microtubules
and PHF formation. Despite the fact that several protein kinases were shown to
promote phosphorylation of tau, it was found that only GSK-3 phosphorylation
directly affected tau ability to promote microtubule self assembly (Hanger et
al.,
1992; Mandelkow et al., 1992; Mulot et al., 1994; Mulot et al., 1995). Further
evidence for the GSK-3 role in this respect came from studies of cells
overexpressing
GSK-3 and from transgenic mice that specifically expressed GSK-3 in brain. In
both
cases GSK-3 led to generation of the PHF like epitope tau (Lucas et al.,
2001).
GSK-3 is further linked with Alzheimer's disease by its role in cell
apoptosis.
The fact that insulin is a survival factor of neurons {Barber et al., 2001 )
and initiates
its anti-apoptotic action through activation of PI3 kinase and PKB (Barber et
al.,
2001), suggested that GSK-3, which is negatively regulated by these signaling
components, promotes neuronal apoptosis. Several studies have indeed confirmed
this view, and showed that GSK-3 is critically important in life and death
decision.
Furthermore, its apoptotic function was shown to be independent of PI3 kinase.
Overexpression of GSK-3 in PC12 cells caused apoptosis (Pap et al., 1998).
Activation of GSK-3 in cerebellar granule neurons mediated migration and cell
death
(Tong et al., 2001). In human neuroblastoma SH-SYSY cells, over expression of
GSK-3 facilitated stauroaporine-induced cell apoptosis (Bijur et al., 2000).
The relation between GSK-3 inhibition and the prevention of cells death has
been further demonstrated by studies showing that expression of Fratl, a GSK-3
(3
inhibitor, was sufficient to rescue neurons from death induced by inhibition
of PI3
kinase {Crowder et al., 2000).
Another implication of GSK-3 was detected in the context of affective
disorders, i.e., bipolar disorders and manic depression. This linkage was
based on the
findings that lithium, a primary mood stabilizer frequently used in bipolar
disease, is a
strong and specific inhibitor of GSK-3 at the therapeutic concentration range
used in
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clinics (Klein et al., 1996; Stambolic et al., 1996; Phiel et al., 2001). This
discovery
has led to a series of studies that were undertaken to determine if lithium
could mimic
loss of GSK-3 activity in cellular processes. Indeed, lithium was shown to
cause
activation of glycogen synthesis (Cheng et al., 1983), stabilization and
accumulation
of (3-catenin (Stambolic et al., 1996), induction of axis duplication in
Xehopus embryo
(Klein et al., 1996), and protection of neuronal death (Bijur et al., 2000).
Valproic
acid, another commonly used mood stabilizer has also been found to be an
effective
GSK-3 inhibitor (Chen et al., 1999). Altogether, these studies indicated that
GSK-3 is
a major in vivo target of lithium and valproic acid and thus has important
implications
1o in novel therapeutic treatment of affective disorders.
On.e mechanism by which lithium and other GSK-3 inhibitors may act to treat
bipolar disorder is to increase the survival of neurons subjected to
aberrantly high
levels of excitation induced by the neurotransmitter, glutamate (Nonaka et
al., 1998).
Glutamate-induced neuronal excitotoxicity is also believed to be a major cause
of
neurodegeneration associated with acute damage, such as in cerebral ischemia,
traumatic brain injury and bacterial infection. Furthermore, it is believed
that
excessive glutamate signaling is a factor in the chronic neuronal damage seen
in
diseases such as Alzheimer's, Huntington's, Parkinson's, AIDS associated
dementia,
amyotrophic lateral sclerosis (AML) and multiple sclerosis (MS) (Thomas,
1995).
Consequently, GSK-3 inhibitors are believed to be a useful treatment in these
and other neurodegenerative disorders. Indeed, dysregulation of GSK-3 activity
has
been recently implicated in several CNS disorders and neurodegenerative
diseases,
including schizophrenia (Beasley et al., 2001; Kozlovsky et al., 2002),
stroke, and
Alzheimer's disease {AD) {Bhat and Budd, 2002; Herriandez et al., 2002; Lucas
et al.,
2001; Mandelkow et al., 1992).
Recent work has further demonstrated that GSK-3 is involved in additional
cellular processes including development (He et al, 1995), oncogenesis
(Rubinfeld et
al, 1996) and protein synthesis (Welsh et al, 1993). Importantly, GSK-3 plays
a
negative role in these pathways. This further suggests that GSK-3 is a
cellular
inhibitor in signaling pathways.
In view of the wide implication of GSK-3 in various signaling pathways,
development of specific inhibitors for GSK-3 is considered both promising and
important regarding various therapeutic interventions as well as basic
research.
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As is mentioned above, some mood stabilizers were found to inhibit GSK-3.
However, while the inhibition of GSK-3 both by lithium chloride (L,iCI) (PCT
International patent application WO 97141854) and by purine inhibitors (PCT
International patent application WO 98/16528) has been reported, these
inhibitors are
5 not specific for GSK-3. In fact, it was shown that these drugs affect
multiple
signaling pathways, and inhibit other cellular targets, such as inositol
monophosphatase (IMpase) and histone deacetylases (Berridge et al., 1989;
Phiel and
Klein, 2001 ).
Similarly, an engineered CAMP response element binding protein (CREB), a
known substrate of GSK-3, has been described (Fiol et al, 1994), along with
other
potential GSK-3 peptide inhibitors (Fiol et al, 1990). However, these
substrates also
only nominally inhibit GSK-3 activity.
Other GSK-3 inhibitors were recently reported. Two structurally related small
molecules SB-216763 and SB-415286 (Glaxo SmithKline Pharmaceutical) that
specifically inhibited GSK-3 were developed and were shown to modulate
glycogen
metabolism and gene transcription as well as to protect against neuronal death
induced by reduction in PI3 kinase activity {Cross et al., 2001; Coghlan et
al., 2000).
Another study indicated that Induribin, the active ingredient of the
traditional Chinese
medicine for chronic myelocytic leukemia, is a GSK-3 inhibitor. However,
Indirubin
also inhibits cyclic-dependent protein kinase-2 (CDK-2) (Damiens et al.,
2001).
These GSK-3 inhibitors are ATP competitive and were identified by high
throughput
screening of chemical libraries. It is generally accepted that a major
drawback of
ATP-competitive inhibitors is their limited specificity (Davies et al., 2000).
A general strategy for developing specific peptide and other GSK-3 inhibitors
is reported in WO 01/49709 and in U.S. Patent Application Publication No.
20020147146, by the present inventor, which are incorporated by reference as
if fully
set forth herein. This general strategy is based on defining the structural
features of a
GSK-3 substrate, and developing GSK-3 inhibitors in accordance with these
features.
However, while these publications delineate these structural features and
teach
various short peptides that efficiently inhibit GSK-3 activity, they fail to
teach the
design and synthesis of small molecules that could serve as GSA-3 inhibitors.
PCT/IL03/01057, by the present inventor, discloses that attaching a
hydrophobic
moiety to a termini of a peptide GSK 3 inhibitor enhances its inhibition
activity.
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However, although peptides are intriguing drug targets, their use is
oftentimes
limited by, for example, biological instability, immunogenicity, poor
capability to
cross biological membranes such as cell membranes and the blood brain barrier
(BBB), and the like.
There is thus a widely recognized need for, and it would be highly
advantageous to have, non-peptidic compounds for inhibiting GSK-3 activity,
devoid
of the above limitations.
SUMMARY OF THE INVENTION
The present inventor has now surprisingly found that compounds which are
designed according to the unique features of the recognition motif of a GSK-3
substrate exhibit substrate competitive inhibition activity toward GSK-3 and
can
therefore be efficiently used in various applications where reducing the
activity of
GSK-3 is beneficial.
Hence, according to one aspect of the present invention there is provided a
compound having a general formula:
/s
A
/ Rz
X ~Y
R~Z ~W~Ra
D
wherein:
~, Y, Z and W are each independently a carbon atom or a nitrogen atom;
A is alkyl or absent;
G~ L~D_
E
B is a negatively charged group having a formula ~~ , wherein L is
selected from the group consisting of a phosphor atom, a sulfur atom, a
silicon atom, a
boron atom and a carbon atom; Q, G and D are each independently selected from
the
group consisting of oxygen and sulfur;; and E is selected from the group
consisting of
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hydroxy, alkoxy, aryloxy, carbonyl, thiocarbonyl, O-carboxy, thiohydroxy,
thioalkoxy
and thioaryloxy or absent;
D is selected from the group consisting of hydrogen, alkyl, trihaloalkyl,
cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, halo,
hydroxy, alkoxy,
aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, cyano,
vitro, azo,
sulfonamide, carbonyl, ketoester, thiocarbonyl, ester, ether, thioether,
thiocarbamate,
urea, thiourea, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-
amide,
N-amide, C-carboxy, O-carboxy, sulfonamide, trihalomethanesulfonamido, guanyl,
guanidine, guanidinoallcyl, amino, aminoalkyl, hydrazine and a hydrophobic
moiety;
and
Rl, R2, R3 and R4 are each independently selected from the group consisting of
hydrogen, a lone pair of electrons, alkyl, trihaloalkyl, cycloalkyl, alkenyl,
alkynyl,
aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy,
thiohydroxy,
thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, cyano, vitro, azo, sulfonamide,
carbonyl,
ketoester, thiocarbonyl, ester, ether, thioether, thiocarbamate, urea,
thiourea, O-
carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amide, N-amide, C-
carboxy, O-carboxy, sulfonamide, trihalomethanesulfonamido, guanyl,
guanylinoalkyl, guanidine, guanidinoalkyl, amino, aminoalkyl, hydrazine, and
an
ammonium ion,
or a pharmaceutically acceptable salt thereof,
provided that at least one of X, Y, Z and W is a nitrogen atom and/or at least
one of Rl, R2, R3 and R4 is a group containing at least one amino moiety, and
with the
proviso that the compound is not pyridoxal phosphate
According to further features in preferred embodiments of the invention
described below, D in the above formula is a hydrophobic moiety, and thus,
according
to another aspect of the present invention there is provided a compound having
the
formula described above, wherein D is a hydrophobic moiety. The compound
according to this aspect of the present invention includes also the compounds
excluded above, substituted by the hydrophobic moiety.
The compounds described hereinabove are capable of inhibiting an activity of
GSK-3.
According to still further features in the described preferred embodiments A
is
alkyl.
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According to still further features in the described preferred embodiments L
is
a phosphor atom.
According to still further features in the described preferred embodiments
each
of Q, G and D is oxygen, and E is preferably hydroxy.
According to still further features in the described preferred embodiments at
least one of X, Y, Z and W is a nitrogen atom.
According to still further features in the described preferred embodiments at
least two of X, Y, Z and W are nitrogen atoms. Preferably either X and Y are
each a
nitrogen atom or Z and W are each a nitrogen atom.
According to still further features in the described preferred embodiments at
least one of Rl, R2, R3 and R~ is a group containing at least one amino
moiety.
According to still further features in the described preferred embodiments at
least two of Rl, R2, R3 and R4 are groups containing at least one amino
moiety.
Preferably either each of Rl and RZ or each of R3 and R4 is a group containing
at least
one amino moiety.
Examples of groups containing at least one amino moiety include, without
limitation, guanidino, guanidinoalkyl, aminoalkyl, analogs thereof,
derivatives thereof
and any combination thereof.
According to still further features in the described preferred embodiments the
group containing at least one amino moiety comprises at least one positively
charged
group
According to still further features in the described preferred embodiments the
positively charged group comprises an ammonium ion. Alternatively, the
positively
charged group has a chemical structure that is derived from a side chain of a
positively charged amino acid, such as, but not limited to, arginine, lysine,
histidine,
proline and any derivative thereof.
When D is hydrophobic moiety, the hydrophobic moiety is preferably selected
from the group consisting of a fatty acid residue, a saturated alkylene chain
having
between 4 and 30 carbon atoms, an unsaturated alkylene chain having between 4
and
30 carbon atoms, an aryl, a cycloalkyl and a hydrophobic peptide sequence.
The fatty acid can be, for example, myristic acid, lauric acid, palmitic acid,
stearic acid, oleic acid, arachidonic acid, linoleic acid or linolenic acid.
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Preferred compounds according to the present invention further include
compounds in which each of X, Y, Z and W is a carbon atom; and at least one of
R~,
R2, R3 and R4 is the group containing at least one amino moiety as described
above.
Further preferred compounds are those in which each of X, Y and Z is a
carbon atom and W is a nitrogen atom.
According to still another aspect of the present invention there is provided a
pharmaceutical composition that comprises, as an active ingredient, a compound
as is
described hereinabove, which is capable of inhibiting an activity of GSK-3,
and a
pharmaceutically acceptable earner.
According to further features in preferred embodiments of the invention
described below, the pharmaceutical composition is packaged in a packaging
material
and is identified in print, on or in the packaging material, for use in the
treatment of a
biological condition associated with GSK-3 activity, as is detailed
hereinbelow.
According to still further features in the described preferred embodiments the
pharmaceutical composition further comprises at least one additional active
ingredient
that is capable of altering an activity of GSK 3, as is detailed hereinbelow.
According to yet another aspect of the present invention there is provided a
method of treating a biological condition associated with an activity of GSK-
3, which
comprises administering to a subject in need thereof a therapeutically
effective
2o amount of a compound that is capable of inhibiting an activity of GSK-3, as
is
described hereinabove.
According to further features in preferred embodiments of the invention
described below, the method according to this aspect of the present invention
further
comprises co-administering to the subject at least one additional active
ingredient,
which is capable of altering an activity of GSK-3.
The additional active ingredient can be an active ingredient that is capable
of
inhibiting an activity of GSK-3 or an active ingredient that is capable of
downregulating an expression of GSK-3.
The biological condition according to the present invention is preferably is
selected from the group consisting of obesity, non-insulin dependent diabetes
mellitus, an insulin-dependent condition, an affective disorder, a
neurodegenerative
disease ar disorder and a psychotic disease or disorder.
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The affective disorder can be a unipolar disorder (e.g., depression) or a
bipolar
disorder (e.g., manic depression).
The neurodegenerative disorder can results from an event selected from the
group consisting of cerebral ischemia, stroke, traumatic brain injury and
bacterial
5 infection, or can be a chronic neurodegenerative disorder that results from
a disease
selected from the group consisting of Alzheimer's disease, Huntington's
disease,
Parkinson's disease, AIDS associated dementia, amyotrophic lateral sclerosis
(AML)
and multiple sclerosis.
According to an additional aspect of the present invention there is provided a
l0 method of inhibiting an activity of GSK-3, which comprises contacting cells
expressing GSK-3 with an inhibitory effective amount of a compound according
to
the present invention.
The activity can be a phosphorylation activity and/or an autophosphorylation
activity.
According to yet an additional aspect of the present invention there is
provided
a method of potentiating insulin signaling, which comprises contacting insulin
responsive cells with an effective amount of the compound described
hereinabove.
In each of these methods, the contacting the cells can be effected in vitro or
in
vivo.
According to further features in preferred embodiments of the invention
described below, each of the methods according to these additional aspects of
the
present invention further comprises contacting the cells with at least one an
additional
active ingredient, as is described hereinabove.
The present invention successfully addresses the shortcomings of the presently
known configurations by providing newly designed, non-peptidic compounds for
inhibiting GSK-3 activity, which can be efficiently used in the treatment of a
variety
of biological conditions.
Unless otherwise defined, all technical and scientific terms used herein have
the same meaning as commonly understood by one of ordinary skill in the art to
which this invention belongs. Although methods and materials similar or
equivalent
to those described herein can be used in the practice or testing . of the
present
invention, suitable methods and materials are described below. In case of
conflict, the
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patent specification, including definitions, will control. In addition, the
materials,
methods, and examples are illustrative only and not intended to be limiting.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention is herein described, by way of example only, with reference to
the accompanying drawings. With specific reference now to the drawings in
detail, it
is stressed that the particulars shown are by way of example and for purposes
of
illustrative discussion of the preferred embodiments of the present invention
only, and
are presented in the cause of providing what is believed to be the most useful
and
readily understood description of the principles and conceptual aspects of the
invention. In this regard, no attempt is made to show structural details of
the
invention in more detail than is necessary for a fundamental understanding of
the
invention, the description taken with the drawings making apparent to those
skilled in
the art how the several forms of the invention may be embodied in practice.
In the drawings:
FIGs. la-b present computer images of the 3D structure of the peptides
p9CREB (Figure 1a) and CREB (Figure lb), as obtained by 2D 1H-NMR studies
(hydrogen atoms not shown; carbon backbone is in gray, nitrogen atoms are in
blue,
oxygen atoms are in red and phosphor atoms are in yellow);
FIG. 2 is an image showing the electrostatic distribution of the p9CREB
peptide, based on the 3D structure of the peptide obtained by 2D 1H-NMR
studies;
FIG. 3 presents the chemical structures of phenyl phosphate, pyridoxal
phosphate (P-5-P), GS-l, GS-2, GS-3 and of the novel compounds GS-4, GS-5 and
GS-21;
FIGs. 4a-b present the 1H NMR spectrum (Figure 4a) and the 13C NMR
spectrum (Figure 4b) of 1,3,5-tris(hydroxylmethyl)benzene, an intermediate in
the
synthesis of GS-21;
FIGS. 5a-b present the 1H NMR spectrum (Figure Sa) and the 13C NMR
spectrum (Figure 5b) of 3,5-Bis(bromomethyl)benzyl alcohol, an intermediate in
the
synthesis of GS-21;
FIGS. 6a-b present the 1H NMR spectrum (Figure 6a) and the 13C NMR
spectrum (Figure 6b) of 3,5-Bis(cyanomethyl)benzyl alcohol, an intermediate in
the
synthesis of GS-21;
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FIG. 7 presents the iH NMR spectrum of 3,5-bis(aminoethyl)benzyl alcohol,
an intermediate in the synthesis of GS-21;
FIG. ~ presents the 1H NMR spectrum of 3,5-bis(te~t-
butoxycarbonylaminoethyl)benzyl alcohol, an intermediate in the synthesis of
GS-21;
FIGS. 9a-c present the iH NMR spectrum (Figure 9a) and the 13C NMR
spectrum (Figure 9b) and the 31P NMR spectrum {Figure 9c) of protected 3,5-
Bis(2-
aminoethyl)benzyl phosphate, an intermediate in the synthesis of GS-21;
FIGS. l0a-d present the 1H NMR spectrum (Figure l0a) and the 13C NMR
spectrum (Figure lOb), the 31P NMR spectrum (Figure lOc) and the ESI-MS
(Figure
lOd) of the TFA salt 3,5-Bis(2-aminoethyl)benzyl phosphate (GS-21 TFA salt);
FIGS. lla-a present the 1H NMR spectrum (Figure lla) and the 13C NMR
spectrum (Figure l lb), the 31P NMR spectrum (Figure 1 lc), the ESI-MS (Figure
1 ld)
and an HPLC chromatogram (Figure l le) of 3,5-Bis(2-aminoethyl)benzyl
phosphate
(GS-21);
FIG. 12 presents comparative plots demonstrating the GSK-3 inhibition
activity of phenyl phosphate, GS-1, GS-2, GS-3 and pyridoxal phosphate (P-5-P)
in in
vitro inhibition assays;
FIG. 13 presents comparative plots demonstrating the GSK-3 inhibition
activity of GS-l, GS-2, GS-3, GS-5 and GS-21 in in vitro inhibition assays
(Black
circles denote GS-1, red circles denote GS-2, green circles denote GS-3, blue
circles
denote GS-5 and pink circles denote GS-21); and
FIGS. 14a b are bar graphs demonstrating the effect of GS-21 (Figure 14b) and
GS-5 (Figure 14a) on glucose uptake in mouse adipocytes, represented by the
j3H] 2-
deoxy glucose incorporation in cells treated with GS-5 and GS-21 as fold
activation
over cells treated with a peptide control (normalized to 1 unit).
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention is of novel, non-peptidic compounds, which are capable
of inhibiting GSK 3 activity and can therefore be used in the treatment of
biological
conditions mediated by GSK-3. Specifically, the present invention is of (i)
compounds that. are designed according to the stearic coordinates of a GSK-3
substrate, which may optionally have a hydrophobic moiety attached thereto;
(ii)
pharmaceutical compositions containing same; (iii) methods of using same for
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13
inhibiting GSK-3 activity and potentiating insulin signaling; and (iv) methods
of
using same in the treatment of biological conditions such as, but not limited
to,
obesity, non-insulin dependent diabetes mellitus, insulin-dependent
conditions,
affective disorders, neurodegenerative diseases and disorders and psychotic
diseases
or disorders.
The principles and operation of the present invention may be better understood
with reference to the drawings and accompanying descriptions.
Before explaining at least one embodiment of the invention in detail, it is to
be
understood that the invention is not limited in its application to the details
set forth in
the following description or exemplified by the Examples. The invention is
capable
of other embodiments or of being practiced or carried out in various ways.
Also, it is
to be understood that the phraseology and terminology employed herein is for
the
purpose of description and should not be regarded as limiting.
One of the parameters that are responsible for substrate-kinase recognition is
an element located within the substrate, which is usually related to as a
"recognition
motif '. As is discussed hereinabove, GSK-3, unlike other kinases, has a
unique
recognition motif, which includes the amino acid sequence SX1XZX3S(p), set
forth in
SEQ ID NO:1, where S is serine or threonine, each of X1, XZ and X3 is any
amino
acid, and S(p) is phosphorylated serine or phosphorylated threonine.
As is widely taught in WO 01/49709 and in U.S. Patent Application
publication No. 20020147146, which are incorporated by reference as if fully
set forth
herein, a set of short peptides which were designed and synthesized based on
this
recognition motif were tested for their activity either as substrates or as
inhibitors.
Base on these assays, a number of features which rendered these peptides
active
substrates or inhibitors toward GSK-3, were determined. One of the most
important
features was that the phosphorylated serine or threonine residue in the motif
is
necessary for binding both substrates and inhibitors to GSK-3. These assays
further
demonstrated that some of these peptides were highly potent and specific
inhibitors of
GSK-3. These peptides were defined as substrate competitive inhibitors.
Based on the findings that GSK-3 recognizes only pre-phosphorylated
substrates, namely, substrates that have a phosphorylated serine or threonine
residue,
it was hypothesized that these pre-phosphorylated GSK-3 substrates has a
unique
structure which allows them to interact with the catalytic core of GSK-3. It
was
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14
further hypothesized that determining this unique structure would enable the
development of small molecules that could act as substrate competitive
inhibitors of
GSK-3.
Thus, in a search for small molecules that would mimic the inhibitory activity
of the small GSK=3 peptide inhibitors described hereinabove, while reducing
the
present invention to practice, the three dimensional structure, as well as the
unique -
structural features, of a short phosphorylated peptide substrate have been
determined,
and a number of compounds characterized by these features were tested for
their
activity as GSK-3 inhibitors.
As a representative example of a GSK-3 substrate the short pre-
phosphorylated peptide p9CREB (ILSRRPS(p)YR, SEQ ID NO:2) was selected.
The three-dimensional structures of p9CREB, as well as of the corresponding
non-
phosphorylated peptide CREB (ILSRRPSYR, SEQ ID N0:3) were determined by 2D
NMR, as is detailed in the Examples section that follows.
As shown in Figures la and lb, the phosphorylated p9CREB substrate has a
defined structure in solution (Figure la}, whereby the corresponding non-
phosphorylated peptide CREB does not exhibit any unique structure (Figure lb).
In view of these results it was suggested that the phosphate group in the
phosphorylated peptide imposes a 'loop-like' structure, through a cation-pi
interaction
between tyrosine {Y8) and arginine (R4) {see, Tables 2 and 3), and as a
result, the
phosphorylated serine at the recognition motif is positioned outside the loop.
Such a
"beaded" structure of the substrate renders the phosphorylated serine
accessible to
interact with the substrate binding pocket of the enzyme.
A support for this suggestion was indeed found in the recently published
crystallization data of GSK-3, described by Dajani et al. (2001). The
crystallization
data of Dajani et al. show that the substrate binding site of GSK-3 comprises
three
positively charged residues, . Arg 96, Arg 180, and Lys 205, which interact
with a
phosphate ion.
Figure 2 presents the electrostatic distribution on the 'surface' 'of the
p9CREB
peptide, based on these findings.
While continuing to conceive the present invention, it was deduced from the
findings described hereinabove that a small molecule that would mimic the
structure
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of a GSK-3 substrate such that it would exerts substrate competitive
inhibitory
activity should be designed according to the following features:
The molecule should include a negatively charged group, preferably a
phosphate group;
5 The negatively charged group should not be stearically hindered; and
The negatively charged group should preferably be flanked at least at one side
thereof or at both sides by one or two positively charged groups.
Based on the above, a general formula of potential compounds for inhibiting
GSK-3 activity has been designed. As is described in the Examples section that
10 follows, preliminary experiments that were conducted with a 'first
generation' of
these compounds, namely, compounds having the most simplified structure of
this
formula, demonstrated the capability of these compounds to inhibit GSK-3
activity,
thus providing a preliminary indication of the inlv.bitory potential of
compounds
having such a formula.
15 A more advanced generation of compounds, which includes novel compounds,
was also designed and synthesized based on the above. As is described in the
Examples section that follows, experiments conducted with these compounds
further
demonstrated their capability to inhibit GSK-3 activity and further to enhance
the
glucose uptake in mice adipocytes, thus demonstrating the promising inhibitory
and
therapeutic effect exerted by compounds designed according to such a formula
Hence, according to one aspect of the present invention there is provided a
compound having a general formula:
/s
A
/~2
\x ~ Y
R~Z /w~lta
D
wherein:
X, Y, Z and W are each independently a carbon atom or a nitrogen atom;
A is allcyl or absent;
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16
G~L~D_
E
B is a negatively charged group having a formula ~~ , wherein L is
selected from the group consisting of a phosphor atom, a sulfur atom, a
silicon atom, a
boron atom and a carbon atom; Q, G and D are each independently selected from
the
group consisting of oxygen and sulfur;; and E is selected from the group
consisting of
hydroxy, alkoxy, aryloxy, carbonyl, thiocarbonyl, O-carboxy, thiohydroxy,
thioalkoxy
and thioaryloxy or absent;
D is selected from the group consisting of hydrogen, alkyl, trihaloalkyl,
cyeloalkyl, ~alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, halo,
hydroxy, alkoxy,
aryloxy, thiohydroxy, thioallcoxy, thioaryloxy, sulfinyl, sulfonyl, cyano,
vitro, azo,
sulfonamide, carbonyl, ketoester, thiocarbonyl, ester, ether, thioether,
thiocarbamate,
urea, thiourea, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-
amido,
N-amido, C-carboxy, O-carboxy, sulfonamido, trihalomethanesulfonamido, guanyl,
guanidino, guanidinoalkyl, amino, aminoalkyl and a hydrophobic moiety; and
Ri, R2, R3 and R~ are each independently selected from the group consisting of
hydrogen, a lone pair of electrons, alkyl, trihaloallcyl, cycloalkyl, alkenyl,
alkynyl,
aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy,
thiohydroxy,
thioalkoxy, thioatyloxy, sulfinyl, sulfonyl, cyano, vitro, azo, sulfonamide,
carbonyl,
ketoester, thiocarbonyl, ester, ether, thioether, thiocarbamate, urea,
thiourea, O-
carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, C-
carboxy, O-carboxy, sulfonamido, trihalomethanesulfonamido, guanyl,
guanylinoalkyl, guanidino, guanidinoalkyl, amino, aminoalkyl, hydrazine and an
ammonium ion, or a pharmaceutically acceptable salt thereof.
It will be appreciated by one of skills in the art that the feasibility of
each of
the substituents (e.g., D, G, E, and Rl-R4) to be located at the indicated
positions
depends on the valency and chemical compatibility of the substituent, the
substituted
position and other substituents. Hence, the present invention is aimed at
encompassing all the feasible substituents for any position.
As used herein, the term "allcyl" refers to a saturated aliphatic hydrocarbon
including straight chain and branched chain groups. Preferably, the alkyl
group has 1
to ~.0 carbon atoms. Whenever a numerical range; e.g., "1-20", is stated
herein, it
implies that the group, in this case the alkyl group, may contain 1 carbon
atom, 2
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carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms. More
preferably, the alkyl is a medium size alkyl having 1 to 10 carbon atoms. Most
preferably, unless otherwise indicated, the alkyl is a lower alkyl having 1 to
4 carbon
atoms. The alkyl group may be substituted or unsubstituted. When substituted,
the
substituent group can be, for example, hydroxyalkyl, trihaloalkyl, cycloalkyl,
alkenyl,
alkynyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy,
thiohydroxy,
thioallcoxy, thioaryloxy, sulfinyl, sulfonyl, cyano, vitro, azo, sulfonyl,
sulfinyl,
sulfonamide, ketoester, carbonyl, thiocarbonyl, ester, ether, thioether,
thiocarbamate,
urea, thiourea, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-
amido,
N-amido, C-carboxy, O-carboxy, trihalomethanesulfonamido, guanyl,
guanylinoalkyl,
guanidino, guanidinoalkyl, amino, aminoalkyl, hydrazine and an ammonium ion.
A "cycloalkyl" group refers to an all-carbon monocyclic or fused ring {i.e.,
rings which share an adjacent pair of carbon atoms) group wherein one of more
of the
rings does not have a completely conjugated pi-electron system. Examples,
without
limitation, of cycloalkyl groups are cyclopropane, cyclobutane, cyclopentane,
cyclopentene, cyclohexane, cyclohexadiene, cycloheptane, cycloheptatriene, and
adamantane. A cycloalkyl group may be substituted or unsubstituted. When
substituted, the substituent group can be, for example, alkyl, hydroxyalkyl,
trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic,
halo,
hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl,
suifonyl,
cyano, vitro, azo, sulfonyl, sulfinyl, sulfonamide, ketoester, carbonyl,
thiocarbonyl,
ester, ether, carboxy, thiocarboxy, thioether, thiocarbamate, urea, thiourea,
O-
carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, C-
carboxy, O-carboxy, sulfonamido, trihalomethanesulfonamido, guanyl,
guanylinoallcyl, guanidino, guanidinoallcyl, amino, aminoalkyl, hydrazine and
an
ammonium ion.
A.n "alkenyl" group refers to an alkyl group, as defined hereinabove, which
consists of at least two carbon atoms and at least one carbon-carbon double
bond.
An "alkynyl" group refers to an alkyl group, as defined hereinabove, which
consists of at least two carbon atoms and at least one carbon-carbon triple
bond.
An. "aryl" group refers to an all-carbon monocyclic or fused-ring polycyclic
(i.~., rings which share adjacent pairs of carbon atoms) groups having a
completely
conjugated pi-electron system. Examples, without limitation, of aryl groups
are
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phenyl, naphthalenyl and anthracenyl. The aryl group may be substituted or
unsubstituted. When substituted, the substituent group can be, for example,
allcyl,
hydroxyallcyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl,
heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy,
thioaryloxy,
sulfinyl, sulfonyl, cyano, vitro, azo, sulfonyl, sulfmyl, sulfonamide,
ketoester,
carbonyl, thiocarbonyl, ester, ether, carboxy, thiocarboxy, thioether,
thiocarbamate,
urea, thiourea, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, G-
amido,
N-amido, C-carboxy, O-carboxy, sulfonamido, trihalomethanesulfonamido, guanyl,
guanylinoalkyl, guarvidino, guanidinoalkyl, amino, aminoalkyl, hydrazine and
an
ammonium ion.
A "heteroaryl" group refers to a monocyclic or fused ring {i.e., rings which
share an adjacent pair of atoms) group having in the rings) one or more atoms,
such
as, for example, nitrogen, oxygen and sulfur and, in addition, having a
completely
conjugated pi-electron system. Examples, without limitation, of heteroaryl
groups
include pyrrole, furane, thiophene, imidazole, oxazole, thiazole, pyrazole,
pyridine,
pyrimidine, quinoline, isoquinoline and purine. The heteroaryl group may be
substituted or unsubstituted. When substituted, the substituent group can be,
for
example, alkyl, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl,
aryl,
heteroaryl, heteroalicyciic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy,
thioalkoxy,
thioaryloxy, sulfinyl, sulfonyl, cyano, vitro, azo, sulfonyl, sulfinyl,
sulfonamide,
ketoester, carbonyl, thiocarbonyl, ester, ether, carboxy, thiocarboxy,
thioether,
thiocarbamate, urea, thiourea, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-
thiocarbamyl, G-amido, N-amido, G-carboxy, O-carboxy, sulfonamido,
trihalomethanesulfonamido, guanyl, guanylinoalkyl, guarvidino, guanidinoalkyl,
2s amino, aminoallcyl, hydrazine and an ammonium ion.
A "heteroalicyclic" group refers to a monocyclic or fused ring group having in
the ring{s) one or more atoms such as nitrogen, oxygen and sulfur. The rings
may
also have one or more double bonds. However, the rings do not have a
completely
conjugated pi-electron system. The heteroalicyclic .may be substituted or
uvsubstituted. When substituted, the substituted group can be, for example,
lone pair
electrons, alkyl, hydroxyallcyl, trihaloalkyi, cycloalkyl, allcenyl, alkynyl,
aryl,
heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy,
thioalkoxy,
thioaryloxy, sulfinyl, sulfonyl, cyano, vitro, azo, sulfonyl, sulfinyl,
sulfonamide,
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ketoester, carbonyl, thiocarbonyl, ester, ether, carboxy, thiocarboxy,
thioether,
thiocarbamate, urea, thiourea, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-
thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido,
trihalomethanesulfonamido, guanyl, guanylinoallcyl, guanidino, guanidinoalkyl,
amino, aminoalkyl, hydrazine and an ammonium ion. Representative examples are
piperidine, piperazine, tetrahydrofurane, tetrahydropyrane, morpholino and the
like.
A "lone pair of electrons" refers to a pair of electrons that are not
participating
in a bond. The lone pair of electrons is present only when X, Y, Z or W is an
unsubstituted nitrogen atom.
A "hydroxy" group refers to an -OH group.
An "azo" group refers to a -N=N group. .
An "alkoxy" group refers to both an -O-alkyl and an -O-cycloalkyl group, as
defined herein.
An "aryloxy" group refers to both an -O-aryl and an -O-heteroaryl group, as
defined herein.
A "thiohydroxy" group refers to a -SH group.
A "thioalkoxy" group refers to both an -S-alkyl group, and an -S-cycloalkyl
group, as defined herein.
A "thioaryloxy" group refers to both an -S-aryl and an -S-heteroaryl group, as
defined herein.
A "carbonyl" group refers to a -C{=O)-R' group, where R' is hydrogen, allcyl,
allcenyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) or
heteroalicyclic
{bonded through a ring carbon) as defined herein.
An "aldehyde" group refers to a carbonyl group, where R' iS hydrogen.
A "thiocarbonyl" group refers to a -C{=S)-R' group, where R' is as defined
herein for R'.
A "C-carboxy" group refers to a -C(=O)-O-R' groups, where R' is as defined
herein.
An "O-caxboxy" group refers to an. R' C(=O)-O- group, where R' is as defined
herein.
A "carboxylic acid" group refers to a C-carboxyl group in which R is
hydrogen.
A "halo" group refers to fluorine, chlorine, bromine or iodine.
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A "trihalomethyl" group refers to a -CX3 group wherein X is a halo group as
defined herein.
A "trihalomethanesulfonyl" group refers to an X3CS(=O)2- group wherein X is
a halo group as defined herein.
5 A "sulfinyl" group refers to an -S(=O)-R' group, where R' is as defined
herein.
A "sulfonyl" group refers to an -S(=O)z-R' group, where R' is as defined
herein.
An "S-sulfonamido" group refers to a -S(=O)2-NR'R" group, with R' as
10 defined herein and R" is as defined for R'.
An "N-sulfonamido" group refers to an R'S(=O)2 NR" group, where R' and
R" are as defined herein.
A "trihalomethanesulfonamido" group refers to an X3CS(=O)2NR'- group,
where R' and X are as defined herein.
15 An "O-carbamyl" group refers to an -OC(=O)=NR.'R" group, where R' and
R" are as defined herein.
A "N-carbamyl" group refers to an R"OC(=O)-NR'- group, where R' and R"
are as defined herein.
An "O-thiocarbamyl" group refers to an -OC(=S)-NR'R" group, where R' and
20 R" are as defined herein.
An "N-thiocarbamyl" group refers to an R"OC(=S)NR'- group, where R' and
R" are as defined herein.
An "amino" group refers to an NR'R" group where R' and R" are as defined
herein.
An "aminoalkyl" group refers to an alkyl, as defined hereinabove, substituted
by an amino group. Preferably, the alkyl terminates by the amino group.
A "C-amido" group refers to a -C(=O)-NR'R" group, where R' and R" are as
defined herein.
An "N-amido" group refers to an R'C(=O)-NR" group, where R' and R" are
as defined herein.
A "urea" group refers to an NR'C(=O)-NR"R"' group, where R' and R" are
as defined herein and R"' is defined as either R' or R" .
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A "guanidino" group refers to an -R'NC(--NR" ")-NR"R"' group, where R',
R" and R"' are as defined herein and R"" is defined as either R', R" or R"'.
A "guanidinoalkyl" group refers to an allcyl group substituted by a guanidino
group, as these terms are defined herein. Preferably, the alkyl group
terminates by the
guanidino group.
A "guanyl" group refers to an R'R"NC(=NR" ")- group, where R', R", R"'
and R" " are as defined herein.
A "guanylinoalkyl" group refers to an allcyl group substituted by a guanyl
group, as these terms are defined herein. Preferably, the alkyl group
terminates by the
guanyl group.
A "vitro" group refers to a -N02 group.
A "cyano" group refers to a -C---N group.
The term "ketoester" describes a -C(=O)-C(=O)-O- group.
The term "thiourea" describes a -NR'-C(=S)-NR'- group, with R' and R" as
defined hereinabove.
The term "hydrazine" describes a NR'-NR" group, with R' and R" as defined
hereinabove.
The term "ammonium ion" described a (NR'R"R"')k, where R', R" and R"'
as defined hereinabove.
The compounds of the present invention are therefore based on a rigid
structure, namely an aromatic (where X, Y, Z and W are all carbon atoms) or a
heteroaromatic (where at least one of X, Y, Z and W is a nitrogen atom) ring,
to
which a negatively charged group is attached. As this structure mimics the
unique
structure of a GSK-3 substrate by providing a negatively charged group which
is not
stearically hindered and has a geometrical structure similar or identical to a
phosphate
group, these compounds are capable of inhibiting GSK-3 activity.
The phrases "negatively charged group" and "positively charged group", as
used herein, refer to an ionizable group, which upon ionization, typically in
an
aqueous medium, has at least one negative or positive electrostatic charge,
respectively. The charged groups can be present in the compounds of the
present
invention either in their ionized form or as a pre-ionized form.
The negatively charged group according to the present invention has a
structure as defined hereinabove, whereby the positively charged group can be
a
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positively charged ion per se (e.g., an ammonium ion) or any group (e.g.,
alkyl,
cycloalkyl, aryl, etc.) that is substituted by a positively charged ion (e.g.,
a secondary,
tertiary or quaternary ammonium ion, an ionized aminoalkyl, etc.j.
Preferably, the negatively charged group is a phosphate group, such that in
the
formula above L is a phosphor atom, whereby each of Q, G and D is oxygen.
Further
preferably, E is hydroxy. Alternatively, the hydroxy group can also be ionized
so as
to have another negative electrostatic charge.
Alternatively, the negatively charged group can be a thiophosphate group,
sulfate or sulfonate group, a borate or boronate group and the like, according
to the
l0 formula above.
The negatively charged group is preferably attached to the aryl/heteroaryl
ring
via an alkyl group, such that A in the formula above is alkyl, preferably an
unsubstituted alkyl, and more preferably a methyl.
The attachment of the negatively charged group to the ring via an alkyl group
renders the negatively charged group a free rotatable group as opposed to its
rigidity
when attached directly to the ring. The free rotatability of the negatively
charged
group is advantageous since it enables the negatively charged group to readily
interact
with the binding site of the enzyme.
It should be noted herein that although the direct or indirect attachment of a
phosphate or any other negatively charged groups according to the present
invention
to an aromatic or heteroaromatic ring is effected via simple procedures and
results in
structurally simple compounds, only a limited number of such compounds have
been
synthesized hitherto. These include pyridoxal phosphate, benzyl phosphate,
phenyl
phosphate and a limited number of derivatives thereof (e.g., substituted
pyridoxal
phosphate, benzyl phosphate and phenyl phosphate). It is assumed that since
heretofore no biological activity has been associated with such compounds, one
ordinarily skilled in the art was not motivated to provide such .compounds.
However,
the compounds according to this aspect of the invention exclude any of the
presently
known compounds that are embraced by the formula above.
As is noted hereinabove, the base structure of the compounds of the present
invention is an aromatic ring or a heteroaromatic ring.
However, since it is preferable to have one, and more preferably two,
positively charged groups that flank the negatively charged group, the ring is
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23
preferably a heteroaromatic ring, such that in the formula above at least one
of X, Y,
Z and W is a nitrogen atom. Preferably, Z or W is a nitrogen atom.
Further preferably at least two of X, Y, Z and W are nitrogen atoms, more
preferably either X and Y are nitrogen atoms or Z and W are nitrogen atoms,
and even
more preferably Z and W are nitrogen atoms.
As is well known in the art, a nitrogen atom within an aromatic ring is
typically basic under neutral conditions and therefore, at a biological
environment, it
tends to be protonated so as to produce a positively charged =NH+- group. As
is
described hereinabove, a compound that has one or two of such positively
charged
groups flanking the negatively charged group is preferable.
As an alternative or in addition to the positively charged nitrogen atoms
within
the base ring, preferably at least one of Rl, R2, R3 and R4 is a group
containing at least
one amino moiety.
As used herein, the phrase "group containing at least one amino moiety" refers
to those groups described above (e.g., alkyl, cycloalkyl, aryl, etc.) which
contain one
or more amino moiety, as this term is defined herein.
Representative examples of groups containing at least one amino moiety
include, without limitation, an amino, an aminoalkyl, hydrazine, urea,
thiourea,
guanyl, amido, carbamyl, guanidino, guanidinoalkyl and guanylinoalkyl, as
these
terms are defined herein.
As is well known in the art, a free amino group is typically basic under
neutral
conditions and therefore, at a biological environment, it tends to be
protonated so as to
produce a positively charged NHs~ group, for example. As is described
hereinabove,
a compound that has one or two of such positively charged groups flanking the
negatively charged group is preferable.
Thus, the amino moiety is preferably present in this group as a readily-
protonated moiety, that is a moiety in which the amino nitrogen has a
substantial
partially negative charge.
Preferred examples of groups containing at least one amino moiety therefore
include, without limitation, an amino, an aminoalkyl, hydrazine, guanyl,
guanylinoalkyl, guanidino, guanidinoalkyl and guanylinoalkyl, as these terms
are
defined herein.
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The groups containing at least one amino moiety can be present in the
compounds of the present invention either as is or as positively charged
groups, in
which at least one of the amino moieties is ionized.
As is described above, positively charged groups according to the present
invention comprise an ammonium ion, such that representative examples of
positively
charged groups include, without limitation, an ammonium ion per se {a
protonated
amino group) and any group that bears an ammonium ion, as is defined
hereinabove,
such as an alkyl, cycloalkyl or aryl substituted by an ammonium ion,
guanidino,
guanyl, hydrazine and the like.
Particularly preferred are positively charged groups that have a chemical
structure derived from a side chain. of a positively charged amino acid, e.g.,
lysine,
arginine, histidine, proline and derivatives thereof, with the first two being
the most
preferred.
By "a chemical structure derived from a side chain of a positively charged
amino acid" it is meant that the positively charged group has a similar or
identical
chemical structure as such a side chain.
Preferably either Rl and RZ or R3 and R4 are groups containing at least one
amino moiety (e.g., positively charged groups), which flank the negatively
charged
group, as desired.
Hence, preferred compounds according to the present invention are those
having the following formulas:
0_
~~OH
O
m~2C)~
GHQ ~~i K
wherein m is an integer from 1 to 6; each of Ql and QZ is independently a
carbon atom or a nitrogen atom; and G and/or K are each a group containing a
free
amino moiety (e.g., a positively charged group).
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As is described above and is further demonstrated in the Examples section that
follows, a number of compounds were designed according to the general formula
described above, were successfully synthesized and were found to exert GSK-3
inhibition activity. The chemical structures of these compounds are presented
in
5 Figure 3. The efficacy of these compounds as inhibitors of GSK-3 activity is
presented in Figures 12 and 13, whereby their beneficial effect on glucose
uptake in
mice adipocytes is demonstrated in Figures 14a and 14b.
As is shown in Figures 12 and 13 and in the Examples section that follows,
some of the tested compounds do not have a positively charged group (e.g., GS-
1 and
10 GS-2, yet, these compounds exert inhibitory activity towards GSK-3.
However, as is
further shown in Figures 12 and 13, compounds that have a nitrogen atom within
the
base ring were found to be more active inhibitors, thus indicating a
beneficial effect of
such groups.
Hence, additional preferred compounds according to the present invention are
15 compounds according to the general formula described above, in which each
of X, Y,
Z and W is a carbon atom; and at least one of R3 and R4 is a group containing
an
amino moiety (e.g., a positively charged group); and D is hydrogen or alkyl.
More
preferred compounds are those where each of X, Y and Z is a carbon atom and W
is a
nitrogen atom.
20 In another preferred embodiment of this aspect of the present invention,
the
compound has a hydrophobic moiety attached thereto.
As is described in detail in PCT/IL03/10157, by the present inventor, it was
found that attaching a hydrophobic moiety to the N-terminus of GSK-3 peptide
inhibitors enhances the inhibitory activity of the peptides.
25 Since the phosphorylated residue in the peptide inhibitors is located at
the C-
terminus thereof, it is assumed that compounds according to the present
invention,
which include a hydrophobic moiety that is located at the most distal position
to the
negatively charged group, as in the case of the peptide inhibitors, will exert
enhanced
inhibitory activity.
Hence, according to another aspect of the present invention there is provided
a
compound that has the general formula described hereinabove, where D is a
hydrophobic moiety.
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As used herein the phrase "hydrophobic moiety" refers to any substance or a
residue thereof that is characterized by hydrophobicity.
As is well accepted in the art, the term "residue" describes a major portion
of a
substance that is covalently linked to another substance, herein the compound
described hereinabove.
Hence, a hydrophobic moiety according to the present invention is preferably
a residue of a hydrophobic substance, and is preferably covalently attached to
the
compound described hereinabove.
Representative examples of hydrophobic substances from which the
l0 hydrophobic moiety of the present invention can be derived include, without
limitation, a saturated alkylene chain, an unsaturated alkylene chain., an
aryl, a
cycloalkyl and a hydrophobic peptide sequence.
As used herein, the phrase "alkylene chain" refers to a hydrocarbon linear
chain, which can be saturated or unsaturated. The alkylene chain can be
substituted or
unsubstituted, as is described above with respect to an alkyl group, and can
be fiu-ther
interrupted by one or more heterogamous such as nitrogen, oxygen, sulfur,
phosphor
and the like. The alkylene chain preferably includes at least 4 carbon atoms,
more
preferably at least 8 carbon atoms, more preferably at least 10 carbon atoms
and mat
have up to 20, 25 and even 30 carbon atoms.
The hydrophobic moiety of the present invention can therefore comprise a
residue of the hydrophobic substances described hereinabove.
A preferred example of an alkylene chain according to this aspect of the
present invention is an alkylene chain that comprises a carboxy group, namely,
a fatty
acid residue(s).
Preferred fatty acids that are usable in the context of the present invention
include, without limitation, saturated or unsaturated fatty acids that have
more than 10
carbon atoms, preferably between 12 and 24 carbon atoms, such as, but not
limited to,
myristic acid, lauric acid, palmitic acid, stearic acid, oleic acid, linoleic
acid, linolenic
acid, arachidonic acid etc.
Alternatively, the hydrophobic moiety, according to the present invention, can
be a hydrophobic peptide sequence. The hydrophobic peptide sequence, according
to
the present invention, preferably includes between 2 and 1 S amino acid
residues, more
preferably between 2 and 10 amino acid residues, more preferably between 2 and
5
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27
amino acid residues, in which at least one amino acid residue is a hydrophobic
amino
acid residue.
Representative examples of hydrophobic amino acid residues include, without
limitation, an alanine residue, a cysteine residue, a glycine residue, an
isoleucine
residue, a leucine residue, a valine residue, a phenylalanine residue, a
tyrosine
residue, a methionine residue, a proline residue and a tryptophan residue, or
any
modification thereof, as is described hereinabove.
Alternatively, the hydrophobic amino acid residue can include any other
amino acid residue, which has been modified by incorporation of a hydrophobic
moiety thereto.
As used herein, the phrase "amino acid residue", which is also referred to
herein, interchangeably, as "amino acid", describes an amino acid unit within
a
polypeptide chain. The amino acid residues within the hydrophobic peptide
sequence
can be either natural or modified amino acid residues, as these phrases are
defined
hereinafter.
As used herein, the phrase "natural amino acid residue" describes an amino
acid residue, as this term is defined hereinabove, which includes one of the
twenty
amino acids found in nature.
As used herein, the phrase "modified amino acid residue" describes an amino
acid residue, as this term is defined hereinabove, which includes a natural
amino acid
that was subjected to a modification at its side chain. Such modifications are
well
known in the art and include, for example, incorporation of a functionality
group such
as, but not limited to, a hydroxy group, an amino group, a carboxy group and a
phosphate group within the side chain. This phrase therefore includes, unless
otherwise specifically indicated, chemically modified amino acids, including
amino
acid analogs (such as penicillamine, 3-mercapto-D-valine), naturally-occurring
non-
proteogenic amino acids (such as norleucine), and chemically-synthesized
compounds
that have properties known in the art to be characteristic of an amino acid.
The term
"proteogenic" indicates that the amino acid can be incorporated into a protein
in a cell
through well-known metabolic pathways.
Accordingly, as used herein, the term "amino acid" or "amino acids" is
understood to include the 20 naturally occurring amino acids; those amino
acids often
modified post-translationally in vivo, including, for example, hydroxyproline,
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28 .
phosphoserine and phosphothreonine; and other unusual amino acids including,
but
not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine,
nor-
leucine and ornithine. Furthermore, the term "amino acid" includes both D- and
L-
amino acids which are linked via a peptide bond or a peptide bond analog to at
least
one addition amino acid as this term is defined herein.
As the hydrophobic moiety provides for enhanced unpredictable activity,
known compounds such as phenyl phosphate and pyridoxal phosphate, which are
substituted by a hydrophobic moiety, are also included within the scope of
this aspect
the present invention.
As is discussed hereinabove and is further demonstrated in the Examples
section that follows, the compounds of the present invention, described
hereinabove,
are designed based on the three-dimensional structure of a GSK-3 substrate and
are
therefore potential substrate competitive inhibitors of GSK-3 activity.
Hence, according to still another aspect of the present invention, there is
provided a method of inhibiting an activity of GSK-3, which is effected by
contacting
cells expressing GSK-3 with an inhibitory effective amount of a compound
described
hereinabove.
As used herein, the term "inhibitory effective amount" is the amount
determined by such considerations as are known in the art, which is sufficient
to
inhibit the activity of GSK-3. The activity can be a phosphorylation and/or
autophosphorylation activity of GSK-3.
The method according to this aspect of the present invention can be effected
by contacting the cells with the compounds ih vitro and/or in vivo. This
method can
be further effected by further contacting the cells with an additional active
ingredient
~ that is capable of altering an activity of GSK-3, as is detailed
hereinbelow.
The inhibition of GSK-3 activity is a way to increase insulin activity in
vivo.
high activity of GSK-3 impairs insulin action in intact cells (Eldar-Finkelman
et al,
1997). This impairment results from the phosphorylation of insulin receptor
substrate-1 (IRS-1) serine residues by GSK-3. Studies performed in patients
with
type II diabetes (non-insulin dependent diabetes mellitus, NIDDIVn show that
glycogen synthase activity is markedly decreased in these patients, and that
decreased
activation of protein kinase B (PKB), an upstream. regulator of GSK-3, by
insulin is
also detected (Shulman et al, (1990); Nikoulina et al, (1997); Cross et al,
(1995).
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29
Mice susceptible to high fat diet-induced diabetes and obesity have
significantly
increased GSK-3 activity in epididymal fat tissue (Eldar-Finkeliuan et al,
1999).
Increased GSK-3 activity expressed in cells resulted in suppression of
glycogen
synthase activity (Eldar-Finkelman et al, 1996).
Inhibition of GSK-3 activity therefore provides a useful method for increasing
insulin activity in insulin-dependent conditions. Thus, according to yet
another aspect
of the present invention there is provided a method of potentiating insulin
signaling,
which is effected by contacting insulin responsive cells with an effective
amount, as is
defined hereinabove, of a compound according to the present invention.
As used herein, the phrase "potentiating insulin signaling" includes an
increase in the phosphorylation of insulin receptor downstream components and
an
increase in the rate of glucose uptake as compared with glucose uptake in
untreated
subjects or cells.
The method according to this aspect of the present invention can be effected
by contacting the cells with the compound of the present invention, i~c vitf~o
or i~ vivo,
and can be also effected by further contacting the cells with insulin.
Potentiation of insulin signaling, i~ vivo, resulting from administration of
the
conjugates of the present invention, can be monitored as a clinical endpoint.
In
principle, the easiest way to look at insulin potentiation in a patient is to
perform the
glucose tolerance test. After fasting, glucose is given to a patient and the
rate of the
disappearance of glucose from blood circulation (namely glucose uptake by
cells) is
measured by assays well known in. the art. Slow rate (as compared to healthy
subject)
of glucose clearance will indicate insulin resistance. The administration of
an
inhibitor to an insulin-resistant patient increases the rate of glucose uptake
as
compared with a non-treated patient. The inhibitor may be administered to an
insulin
resistant patient for a longer period of time, and the levels of insulin,
glucose, and
leptin in blood circulation (which are usually high) may be determined.
Decrease in
glucose levels will indicate that the inhibitor potentiated insulin action. A
decrease in
insulin and leptin levels alone may not necessarily indicate potentiation of
insulin
action, but rather will indicate improvement of the disease condition by other
mechanisms.
The compounds of the present invention, described hereinabove, can be
effectively utilized for treating any biological condition that is associated
with GSK-3.
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Hence, according to an additional aspect of the present invention, there is
provided a method of treating a biological condition associated with GSK-3
activity.
The method, according to this aspect of the present invention, is effected by
administering to a subject in need thereof a therapeutically effective amount
of the
5 compound of the present invention, described hereinabove.
The phrase "biological condition associated with GSK-3 activity" as used
herein includes any biological or medical condition or disorder in which
effective
GSK-3 activity is identified, whether at normal or abnormal levels. The
condition or
disorder may be caused by the GSK-3 activity or may simply be characterized by
10 GSK-3 activity. That the condition is associated with GSK-3 activity means
that
some aspect of the condition can be traced to the GSK-3 activity.
Herein, the term "treating" includes abrogating, substantially inhibiting,
slowing or reversing the progression of a condition or disorder, substantially
ameliorating clinical symptoms of a condition or disorder or substantially
preventing
15 the appearance of clinical symptoms of a condition or disorder. These
effects may be
manifested, for example, by a decrease in the rate of glucose uptake with
respect to
type II diabetes or by halting neuronal cell death with respect to
neurodegenerative
disorders, as is detailed hereinbelow.
The term "administering" as used herein describes a method for bringing the
20 compound of the present invention and cells affected by the condition or
disorder
together in such a manner that the compound can affect the GSK-3 activity in
these
cells. The compounds of the present invention can be administered via any
route that
is medically acceptable. The route of administration can depend on the
disease,
condition or injury being treated. Possible administration routes include
injections,
25 by parenteral routes, such as intravascular, intravenous, infra-arterial,
subcutaneous,
intramuscular, intratumor, intraperitoneal, intraventricular, intraepidural,
intracerebrovascular or others, as well as oral, nasal, ophthalmic, rectal,
topical, or by
inhalation. Sustained release administration is also specifically included in
the
invention, by such means as depot injections or erodible implants.
Administration can
30 also be infra-articularly, intrarectally,. intraperitoneally,
intramuscularly,
subcutaneously, or by aerosol inhalant. Where treatment is systemic, the
compound
can be administered orally or parenterally, such as intravenously,
intramuscularly,
subcutaneously, intraorbitally, intracapsularly, intraperitoneally or
intracisternally, as
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long as provided in a composition suitable for effecting the introduction of
the
compound into target cells, as is detailed hereinbelow.
The phrase "therapeutically effective amount", as used herein, describes an
amount administered to an individual, which is sufficient to abrogate,
substantially
inhibit, slow or reverse the progression of a condition associated with GSK-3
activity,
to substantially ameliorate clinical symptoms of a such a condition or
substantially
prevent the appearance of clinical symptoms of such a condition. The GSK-3
activity can be a GSK-3 kinase activity. The inhibitory amount may be
determined
directly by measuring the inhibition of a GSK-3 activity, or, for example,
where the
desired effect is an effect on an activity downstream of GSK-3 activity in a
pathway
that includes GSK-3, the inhibition may be measured by measuring a downstream
effect. Thus, for example where inhibition of GSK-3 results in the arrest of
phosphorylation of glycogen synthase, the effects of the compound may include
effects on an insulin-dependent or insulin-related pathway, and the compound
may be
administered to the point where glucose uptake is increased to optimal levels.
Also,
where the inhibition of GSK-3 results in the absence of phosphorylation of a
protein
that is required for further biological activity, for example, the tau
protein, then the
compound may be administered until polymerization of phosphorylated tau
protein is
substantially arrested. Therefore, the inhibition of GSK-3 activity will
depend in part
on the nature of the inhibited pathway or process that involves GSK-3
activity, and on
the effects that inhibition of GSK-3 activity has in a given biological
context.
The amount of the compound that will constitute an inhibitory amount will
vary depending on such parameters as the compound and its potency, the half
life of
the compound in the body, the rate of progression of the disease or biological
condition being treated, the responsiveness of the condition to the dose of
treatment or
pattern of administration, the formulation, the attending physician's
assessment of the
medical situation, and other relevant factors, and in general the health of
the patient,
and other considerations such as prior administration of other therapeutics,
or co-
administration of any therapeutic that will have an effect on the inhibitory
activity of
the compound or that will have an effect on GSK-3 activity, or a pathway
mediated by
GSK-3 activity. It is expected that the inhibitory amount will fall in a
relatively
broad range that can be determined through routine trials.
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32
As is discussed in detail hereinabove, GSK-3 is involved in various biological
pathways and hence, the method according to this aspect of the present
invention can
be used in the treatment of a variety of biological conditions, as is detailed
hereinunder.
GSK-3 is involved in the insulin signaling pathway and therefore, in one
example, the method according this aspect of the present invention can be used
to
treat any insulin-dependent condition.
As GSK-3 inhibitors are known to inhibit differentiation of pre-adipocytes
into
adipocytes, in another example, the method of this aspect of the present
invention can
be used to treat obesity.
In yet another example, the method according to this aspect of the present
invention can be used to treat diabetes and particularly, non-insulin
dependent
diabetes mellitus.
Diabetes mellitus is a heterogeneous primary disorder of carbohydrate
metabolism with multiple etiologic factors that generally involve insulin
deficiency or
insulin resistance or both. Type I, juvenile onset, insulin-dependent diabetes
mellitus,
is present in patients with little or no endogenous insulin secretory
capacity. These
patients develop extreme hyperglycemia and are entirely dependent on exogenous
insulin therapy for immediate survival. Type II, or adult onset, or non-
insulin-
dependent diabetes mellitus, occurs in patients who retain some endogenous
insulin
secretory capacity, but the great majority of them are both insulin deficient
and insulin
resistant. Approximately 95 % of all diabetic patients in the United States
have non-
insulin dependent, Type II diabetes mellitus (NIDDM), and, therefore, this is
the form
of diabetes that accounts for the great majority of medical problems. Insulin
resistance is an underlying characteristic feature of NIDDM and this metabolic
defect
leads to the diabetic syndrome. Insulin resistance can be due to insufficient
insulin
receptor expression, reduced insulin-binding affinity, or any abnormality at
any step
along the insulin signaling pathway (see U.S. Patent No. 5,861,266).
The compounds of the present invention can be used to treat type II diabetes
in
a patient with type II diabetes as follows: a therapeutically effective amount
of the
compound is administered to the patient, and clinical markers, e.g., blood
sugar level,
are monitored. The compounds of the present invention can further be used to
prevent type II diabetes in a subject as follows: a prophylactically effective
amount of
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33
the compound is administered to the patient, and a clinical marker, for
example IRS-1
phosphorylation, is monitored.
Treatment of diabetes is determined by standard medical methods. A goal of
diabetes treatment is to bring sugar levels down to as close to normal as is
safely
possible. Commonly set goals are 80-120 milligrams per deciliter (mgldl)
before
meals and 100-140 mgldl at bedtime. A particular physician may set different
targets
for the patent, depending on other factors, such as how often the patient has
low blood
sugar reactions. Useful medical tests include tests on the patient's blood and
urine to
determine blood sugar level, tests for glycated hemoglobin level {HbAI~; a
measure of
i0 average blood glucose levels over the past 2-3 months, normal range being 4-
6 %),
tests for cholesterol and fat levels, and tests for urine protein level. Such
tests are
standard tests known. to those of skill. in the art (see, for example,
American Diabetes
Association, 1998). A successful treatment program can also be determined by
having fewer patients in the program with diabetic eye disease, kidney
disease, or
nerve disease.
Hence, in one particular embodiment of the method according to this aspect of
the present invention, there is provided a method of treating non-insulin
dependent
diabetes mellitus: a patient is diagnosed in the early stages of non-insulin
dependent
diabetes mellitus. A compound of the present invention is formulated in an
enteric
capsule. The patient is directed to take one tablet after each meal for the
purpose of
stimulating the insulin signaling pathway, and thereby controlling glucose
metabolism
to levels that obviate the need for administration of exogenous insulin
As is further discussed hereinabove, and has been demonstrated in the PCT
International Patent Application entitled "Glycogen Synthase Kinase-3
Inhibitors", by
the same applicant, which is filed on the same date as the instant
application, GSK-3
inhibition is associated with affective disorders. Therefore, in another
example, the
method according to this aspect of the present invention can be used to treat
affective
disorders such as unipolar disorders (e.g., depression) and bipolar disorders
(e.g.,
manic depression).
As GSK-3 is also considered to be an important player in the pathogenesis of
neurodegenerative disorders and diseases, the method according to this aspect
of the .
present invention can be further used to treat a variety of such disorders and
diseases.
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34
In one example, since inhibition of GSK-3 results in halting neuronal cell
death, the method according to this aspect of the present invention can be
used to treat
a neurodegenerative disorder that results from an event that cause neuronal
cell death.
Such an event can be, for example, cerebral ischemia, stroke, traumatic brain
injury or
bacterial infection.
In another example, since GSK 3 activity is implicated in various central
nervous system disorders and neurodegenerative diseases, the method according
to
this aspect of the present invention can be used to treat various chronic
neurodegenerative diseases such as, but not limited to, Alzheimer's disease,
Huntington's disease, Parkinson's disease, AmS associated dementia,
amyotrophic
lateral sclerosis (A.ML) and multiple sclerosis.
As is discussed hereinabove, GSK-3 activity has particularly been implicated
in the pathogenesis of Alzheimer's disease. Hence, in one representative
embodiment
of the method according to this aspect of the present invention, there is
provided a
method of treating a patient with Alzheimer's disease: A patient diagnosed
with
Alzheimer's disease is administered with a compound of the present invention,
which
inhibits GSK-3-mediated tau hyperphosphorylation, prepared in a formulation
that
crosses the blood brain barrier (BBB). The patient is monitored for tau
phosphorylated polymers by periodic analysis of proteins isolated from the
patient's
2o brain cells for the presence of phosphorylated forms of tau on an SDS-PAGE
gel
known to characterize the presence of and progression of the disease. The
dosage of
the compound is adjusted as necessary to reduce the presence of the
phosphorylated
forms of tau protein.
GSK-3 has also been implicated with respect to psychotic disorders such as
schizophrenia, and therefore the method according to this aspect of the
present
invention can be further used to treat psychotic diseases or disorders, such
as
schizophrenia.
The method according to this aspect of the present invention can be further
effected by co-administering to the subject one or more additional active
ingredient{s)
3o which is capable of altering an activity of GSK-3.
As used herein, "co-administering" describes administration of a compound
according to the present invention in combination with the additional active
ingredients) {also referred to herein as active or therapeutic agent). The
additional
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active agent can be any therapeutic agent useful for treatment of the
patient's
condition. The co-administration may be simultaneous, for example, by
administering a mixture of the compound and the therapeutic agents, or may be
accomplished by administration of the compound and the active agents
separately,
5 such as within a short time period. Co-administration also includes
successive
administration of the compound and one or more of another therapeutic agent.
The
additional therapeutic agent or agents may be administered before or after the
compound. Dosage treatment may be a single dose schedule or a multiple dose
schedule.
l0 The additional active ingredient can be insulin.
Preferably, the additional active ingredient is capable of inhibiting an
activity
of GSK-3, such that the additional active ingredient according to the present
invention
can be any GSK-3 inhibitor other than the compounds of the present invention,
e.g., a
short peptide GSK-3 inhibitor as described in WO 01/49709, PCT/IL03/01057 and
15 U.S. Patent Application Publication No. 20020147146A1. Alternatively, the
GSK-3
inhibitor can be, for example, lithium, valproic acid and/or lithium ion.
Alternatively, the additional active ingredient can be an active ingredient
that
is capable of downregulating an expression of GSK-3.
An agent that downregulates GSK-3 expression refers to any agent which
20 affects GSK-3 synthesis (decelerates) or degradation (accelerates) either
at the level
of the mRNA or at the level of the protein. For example, a small interfering
polynucleotide molecule which is designed to down regulate the expression of
GSK-3
can be used as an additional active ingredient according to this embodiment of
the
present invention.
25 An example for a small interfering polynucleotide molecule which can down-
regulate the expression of GSK-3 is a small interfering RNA or siRNA, such as,
for
example, the morpholino antisense oligonucleotides described by in Munshi et
al.
(Munshi CB, GraefF R, Lee HC, J Biol Chem 2002 Dec 20;277(51 ):49453-8), which
includes duplex oligonucleotides which direct sequence specific degradation of
30 mRNA through the previously described mechanism of RNA interference (RNAi)
(Hutvagner and Zamore (2002) Curr. Opin. Genetics and Development 12:225-232).
As used herein, the phrase "duplex oligonucleotide" refers to an
oligonucleotide structure or mimetics thereof, which is formed by either a
single self
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36
complementary nucleic acid strand or by at least two complementary nucleic
acid
strands. The "duplex oligonucleotide" of the present invention can be composed
of
double-stranded RNA (dsRNA), a DNA-RNA hybrid, single-stranded RNA (ssRNA),
isolated RNA (i.e., partially purified RNA, essentially pure RNA), synthetic
RNA and
recombinantly produced RNA.
Preferably, the specific small interfering duplex oligonucleotide of the
present
invention is an oligoribonucleotide composed mainly of ribonucleic acids.
Instructions for generation of duplex oligonucleotides capable of mediating
RNA interference are provided in wv~.~.v.a~nbion..con~.
Hence, the small interfering polynucleotide molecule according to the present
invention can be an RNAi molecule (RNA interference molecule).
Alternatively, a small interfering polynucleotide molecule can be an
oligonucleotide such as a GSK-3-specific antisense molecule or a rybozyme
molecule, further described hereinunder.
Antisense molecules are oligonucleotides, which contain two or more
chemically distinct regions, each made up of at least one nucleotide. These
oligonucleotides typically contain at least one region wherein the
oligonucleotide is
modified so as to confer upon the oligonucleotide increased resistance to
nuclease
degradation, increased cellular uptake, andlor increased binding affinity for
the target
polynucleotide. An additional region of the oligonucleotide may serve as a
substrate
for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. An example for
such includes RNase H, which is a cellular endonuclease which cleaves the RNA
strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in
cleavage
of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide
inhibition of gene expression. Consequently, comparable results can often be
obtained with shorter oligonucleotides when chimeric oligonucleotides are
used,
compared to phosphorothioate deoxyoligonucleotides hybridizing to the same
target
region. Cleavage of the RNA target can be routinely detected by gel
electrophoresis
and, if necessary, associated nucleic acid hybridization techniques known in
the art.
The antisense molecules of the present invention may be formed as composite
structures of two or more oligonucleotides, modified oligonucleotides, as
described
above. Representative U.S. patents that teach the preparation of such hybrid
structures include, but are not limited to, U.S. Pat. Nos. 5,013,30;
5,149,797;
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37
5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065;
5,652,355; 5,652,356; and 5,700,922, each of which is herein fully
incorporated by
reference.
Rybozyme molecules are being increasingly used for the sequence-specific
inhibition of gene expression by the cleavage of mRNAs. Several rybozyme
sequences can be fused to the oligonucleotides of the present invention. These
sequences include but are not limited ANGIOZYME specifically inhibiting
formation
of the VEGF-R (Vascular Endothelial Growth Factor receptor), a key component
in
the angiogenesis pathway, and HEPTAZYME, a rybozyme designed to selectively
destroy Hepatitis C Virus (HCV) RNA, (Rybozyme Pharmaceuticals, Incorporated -
WEB home page).
Further alternatively, a small interfering polynucleotide molecule, according
to
the present invention can be a DNAzyme.
DNAzymes are single-stranded catalytic nucleic acid molecules. A general
model (the "10-23" model) for the DNAzyme has been proposed. "10-23"
DNAzymes have a catalytic domain of 1 S deoxyribonucleotides, flanked by two
substrate-recognition domains of seven to nine deoxyribonucleotides each. This
type
of DNAzyme can effectively cleave its substrate RNA at purine:pyrimidine
junctions
(Santoro, S.W. & Joyce, G.F. Proc. Natl, Acad. Sci. USA 199; for rev of
DNAzymes
see Khachigian, LM Curr Opin Mol Ther 2002;4:119-21).
Examples of construction and amplification of synthetic, engineered
DNAzymes recognizing single and double-stranded target cleavage sites have
been
disclosed in U.S. Pat. No. 6,326,174 to Joyce et al. DNAzymes of similar
design
directed against the human Urokinase receptor were recently observed to
inhibit
Urokinase receptor expression, and successfully inhibit colon cancer cell
metastasis in
vivo (Itoh et al., 20002, Abstract 409, Ann Meeting Am Soc Gen Ther
wcvw.as t.or ). In another application, DNAzymes complementary to bcr-abl
oncogenes were successful in inhibiting the oncogenes expression in leukemia
cells,
and lessening relapse rates in autologous bone marrow transplant in cases of
CML
and ALL.
Oligonucleotides designed according to the teachings of the present invention
can be generated according to any oligonucleotide synthesis method known in
the art
such as enzymatic synthesis or solid phase synthesis. Equipment and reagents
for
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3~
executing solid-phase synthesis are commercially available from, for example,
Applied Biosystems. Any other means for such synthesis may also be employed;
the
actual synthesis of the oligonucleotides is well within the capabilities of
one skilled in
the art.
While being potent therapeutic agents, and since therapeutic applications
often
require administration of effective amounts of an active ingredient to a
treated
individual, the compounds of the present invention are preferably included, as
active
ingredients, in a pharmaceutical composition which further comprises a
pharmaceutically acceptable carrier for facilitating administration of a
compound to
the treated individual and possibly to facilitate entry of the active
ingredient into the
targeted tissues or cells.
Hence, according to still an additional aspect of the present invention there
is
provided a pharmaceutical composition which comprises, as an active
ingredient, a
compound according to the present invention and a pharmaceutically acceptable
carrier.
Hereinafter, the phrases "pharmaceutically acceptable carrier" and
"physiologically acceptable earner" refer to a carrier or a diluent that does
not cause
significant irritation to a subject and does not abrogate the biological
activity and
properties of the administered compound. Examples, without limitations, of
carriers
are propylene glycol, saline, emulsions and mixtures of organic solvents with
water.
Herein the term "excipient" refers to an inert substance added to a
pharmaceutical composition to further facilitate administration of a compound.
Examples, without limitation, of excipients include calcium carbonate, calcium
phosphate, various sugars and types of starch, cellulose derivatives, gelatin,
vegetable
oils and polyethylene glycols.
°The pharmaceutical acceptable earner can further include other agents
such as,
but not limited to, absorption delaying agents, antibacterial agents,
antifungal agents,
antioxidant agents, binding agents, buffering agents, bullring agents,
cationic lipid
agents, coloring agents, diluents, disintegrants, dispersion agents,
emulsifying agents,
excipients, flavoring agents, glidants, isotonic agents, liposomes,
microcapsules,
solvents, sweetening agents, . viscosity modifying agents, wetting agents, and
skin
penetration enhancers.
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39
Techniques for formulation and administration of drugs may be found in
"Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest
edition, which is incorporated herein by reference.
Suitable routes of administration may, for example, include oral,- rectal,
transmucosal, transdermal, intestinal or parenteral delivery, including
intramuscular,
subcutaneous . and intramedullary injections as well as intrathecal, direct
intraventricular, intravenous, intraperitoneal, intranasal, or intraocular
injections.
Pharmaceutical compositions of the present invention may be manufactured
by processes well known in the art, e.g., by means of conventional mixing,
dissolving, .
granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping
or
lyophilizing processes.
Pharmaceutical compositions for use in accordance with the present invention
thus may be formulated in conventional manner using one or more
pharmaceutically
acceptable carriers comprising excipients and auxiliaries, which facilitate
processing
of the compound into preparations which can be used pharmaceutically. The
composition can be formulated in a delivery form such as an aerosol delivery
form,
aqueous solution, bolus, capsule, colloid, delayed release, depot, dissolvable
powder,
drops, emulsion, erodible implant, gel, gel capsule, granules, injectable
solution,
ingestible solution, inhalable solution, lotion, oil solution, pill,
suppository, salve,
suspension, sustained release, syrup, tablet, tincture, topical cream,
transdermal
delivery form. Proper formulation is dependent upon the route of
administration
chosen.
For injection, the compound of the invention may be formulated in aqueous
solutions, preferably in physiologically compatible buffers such as Hank's
solution,
Ringer's solution, or physiological saline buffer with or without organic
solvents such
as propylene glycol, polyethylene glycol. For transmucosal administration,
penetrants
are used in the formulation. Such penetrants are generally known in the art.
For oral administration, the compound can be formulated readily by
combining the compound with pharmaceutically acceptable carriers well known in
the
art. Such carriers enable the compound of the invention to be formulated as
tablets,
pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and
the like, for
oral ingestion by a patient. Pharmacological preparations for oral use can be
made
using a solid excipient, optionally grinding the resulting mixture, and
processing the
CA 02530111 2005-12-20
WO 2005/000192 PCT/IL2004/000570
mixture of granules, after adding suitable auxiliaries if desired, to obtain
tablets or
dragee cores. Suitable excipients are, in particular, fillers such as sugars,
including
lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for
example,
maize starch, wheat starch, rice starch, potato starch, gelatin, gum
tragacanth, methyl
5 cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose and/or
physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If
desired,
disintegrating agents may be added, such as cross-linked polyvinyl
pyrrolidone, agar,
or alginic acid or a salt thereof such as sodium alginate.
Dragee cores are provided with suitable coatings. For this purpose,
l0 concentrated sugar solutions may be used which may optionally contain gum
arabic,
talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium
dioxide,
lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs
or
pigments may be added to the tablets or dragee coatings for identification or
to
characterize different combinations of active ingredient doses.
15 Pharmaceutical compositions, which can be used orally, include push-fit
capsules made of gelatin as well as soft, sealed capsules made of gelatin and
a
plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain
the active
ingredients in admixture with filler such as lactose, binders such as
starches,
lubricants such as talc or magnesium stearate and, optionally, stabilizers. In
soft
20 capsules, the active ingredients may be dissolved or suspended in suitable
liquids,
such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In
addition,
stabilizers may be added. All formulations for oral administration should be
in
dosages suitable for the chosen route of administration.
For buccal administration, the compositions may take the form of tablets or
25 lozenges formulated in conventional manner.
For administration by inhalation, the compound according to the present
invention is conveniently delivered in the form of an aerosol spray
presentation from
a pressurized pack or a nebulizer with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or
30 carbon dioxide. In the case of a pressurized aerosol, the dosage unit may
be
determined by providing a valve to deliver a metered amount. Capsules and
cartridges of, e.g., gelatin for use in , an inhaler or insufflator may be
formulated
CA 02530111 2005-12-20
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41
containing a powder mix of the ingredient and a suitable powder base such as
lactose
or starch.
The compound described herein may be formulated for parenteral
administration, e.g., by bolus injection or continuous infusion. Formulations
for
injection may be presented in unit dosage form, e.g., in ampoules or in
multidose
containers with optionally, an added preservative. The compositions may be
suspensions, solutions or emulsions in oily or aqueous vehicles, and may
contain
formulatory agents such as suspending, stabilizing and/or dispersing agents.
Pharmaceutical compositions for parenteral administration include aqueous
solutions of the compound in water-soluble form. Additionally, suspensions of
the
compound may be prepared as appropriate oily injection suspensions. Suitable
lipophilic solvents or vehicles include fatty oils such as sesame oil, or
synthetic fatty
acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous
injection
suspensions may contain substances, which increase the viscosity of the
suspension,
such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the
suspension may also contain suitable stabilizers or agents which increase the
solubility of the active ingredient to allow for the preparation of highly
concentrated
solutions.
Alternatively, the compound may be in powder form for constitution with a
suitable vehicle, e.g., sterile, pyrogen-free water, before use.
The compound of the present invention may also be formulated in rectal
compositions such as suppositories or retention enemas, using, e.g.,
conventional
suppository bases such as cocoa butter or other glycerides.
The pharmaceutical compositions herein described may also comprise suitable
solid of gel phase carriers or excipients. Examples of such carriers or
excipients
include, but are not limited to, calcium carbonate, calcium phosphate, various
sugars,
starches, cellulose derivatives, gelatin and polymers such as polyethylene
glycols.
Pharmaceutical compositions suitable for use in context of the present
invention include compositions wherein the compound is contained in an amount
effective to achieve the intended purpose. More specifically, a
therapeutically
effective amount means an amount of a compound effective to affect symptoms of
a
condition or prolong the survival of the subject being treated.
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42
Determination of a therapeutically effective amount is well within the
capability of those skilled in the art, especially in light of the detailed
disclosure
provided herein.
For any active ingredient used in the methods of the invention, the
therapeutically effective amount or dose can be estimated initially from
activity
assays in cell cultures and/or animals. Such information can be used to more
accurately determine useful doses in humans.
The dosage may vary depending upon the dosage form employed and the route
of administration utilized. The exact formulation, route of administration and
dosage
can be chosen by the individual physician in view of the patient's condition.
{See e.g.,
Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1
p.l).
Compositions of the present invention may, if desired, be presented in a pack
or dispenser device, such as a FDA approved kit, which may contain one or more
unit
dosage forms containing the compound. The pack may, for example, comprise
metal
or plastic foil, such as a blister pack. The pack or dispenser device may be
accompanied by instructions for administration. The pack or dispenser may also
be
accompanied by a notice associated with the container in a form prescribed by
a
governmental agency regulating the manufacture, use or sale of
pharmaceuticals,
which notice is reflective of approval by the agency of the form of the
compositions
or human or veterinary administration. Such notice, for example, may be of
labeling
approved by the U.S. Food and Drug Administration for prescription drugs or of
an
approved product insert. Compositions comprising a compound of the invention
formulated in a compatible pharmaceutical carrier may also be prepared, placed
in an
appropriate container, and labeled for treatment of an indicated condition.
Suitable
conditions indicated on the label may include, for example, any of the
biological
conditions associated with GSK-3 activity listed hereinabove.
Hence, the pharmaceutical composition of the present invention can be
packaged in a packaging material and identified in print, on or in the
packaging
material, for use in the treatment or prevention of a biological condition
associated
with GSI~-3.
The pharmaceutical composition of the present invention can further
comprises an additional active ingredient that is capable of interfering with
an activity
of GSI~-3, as is described fiereinabove.
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43
Additional objects, advantages, and novel features of the present invention
will become apparent to one ordinarily skilled in the art upon examination of
the
following examples, which are not intended to be limiting. Additionally, each
of the
various embodiments and aspects of the present invention as delineated
hereinabove
and as claimed in the claims section below fords experimental support in the
following examples.
EXAMPLES
Reference is now made to the following examples, which together with the
l0 above descriptions, illustrate the invention in a non limiting fashion.
MATERL4LS AND EXPERIMENTAL METHODS
Materials:
Peptides were synthesized by Genemed Synthesis Inc. (San Francisco, CA).
Radioactive materials were purchased from Amersham Ltd.
Phenyl phosphate and pyridoxal phosphate (also referred to herein as P-5-P)
were obtained from Sigma (Israel).
All reagents and solvents were obtained from commercial sources (e.g.,
' 20 Sigma, Acros, Aldrich) and were used as supplied, unless otherwise
indicated.
GS-l, GS-2 and GS-3 were synthesized according to procedures known in the
art, as is detailed hereinunder.
Syntheses of the novel compounds GS-4, GS-5 and GS-21 were designed and
practiced as described hereinbelow.
Determinatlo~a of a 3D structure of a GSA 3 substrate by NMR Spectroscopy
and Str~uctur~e Calculations:
A small phosphorylated peptide patterned after the known GSK-3 substrate
CREB, denoted p9CREB, and two additional peptides, a non-phosphorylated
peptide,
9CREB, and a variant where S1 was replaced with glutamic acid (which is
thought to
mimic a charged group), 9ECREB, were used in these studies and are listed in
Table 1
below. Time course analyses of peptide phosphorylation by GSK-3 confirmed that
only the phosphorylated peptide, p9CREB, was a substrate for GSK-3, while
9CREB
and 9ECREB completely failed to be phosphorylated by GSK-3 (data not shown),
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44
thus indicating again that phosphorylated serine is an absolute requirement
for GSK
3.
Table 1
Peptide Sequence SEQ ID NO:
p9CREB ILSRRPS(p)YR 2
9CREB ILSRRPSYR 3
9ECREB ILSRRPEYR 4
The 3D structure of p9CREB (Figure la), 9CREB (Figure lb) and 9ECREB
(not shown) by 2D 1H NMR spectroscopy was determined using the following
procedures:
For the structural studies, a solution of each peptide was prepared by
dissolving lyophilized powder in water containing 10 % DZO. 2D-NMR spectra
were
acquired at the 1H proton frequency of 600.13 MHz on a Broker Avance DMX
spectrometer. The carrier frequency was set on the water signal and it was
suppressed
by applying either a WATERGATE method and by low-power irradiation during the
relaxation period. The experimental temperature (280 K) was optimized in order
to
reduce population averaging due to the fast exchange at more ambient
temperatures,
while preserving the best possible spectral resolution. All experiments were
carried
out in the phase sensitive mode (TPPI or States-TPPI) and recorded with a
spectral
width of 12 ppm, with 4K real t2 data points and 512 tl-increments. Two-
dimensional
homonuclear data collected included TOCSY using a MLEV pulse sequence with a
mixing time of 150 msec, and NOESY experiments with mixing times ranging
between 100 and 750 msecs. Typically, the relaxation delays were 1.5 and 2.0
sec in
TOCSY and NOESY experiments, respectively. In the ROESY measurements, the
duration of the spin-lock was set to 400 msec with a power of 3.4 KHz. All
spectra
were calibrated versus tetramethylsilane.
The data was processed using Broker XVVINNMR software (Broker
Analytische Messtechnik, GmbH, version 2.7). All data processing, calculations
and
analysis were done on Silicon Graphics workstations {1NDY 84000 and INDIG02
810000). Zero filling of the indirect dimension and apodization of the free
induction
decay by a shifted squared-sine window function on both dimensions were
applied
prior to Fourier transformation to enhance spectral resolution. The spectra
were
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WO 2005/000192 PCT/IL2004/000570
further phase-corrected by applying an automatic polynomial baseline
correction
developed by Broker.
Resonance assignment was based on the TOCSY and NOESY spectra
measured at the same experimental conditions, according to the sequential
assignment
5 methodology developed by Wuthrich using the Broker software program AURELIA
(Broker Analytic GmbH, version 2.7).
The NOE distance restraints were derived from NOESY spectra recorded at
450 msec. This optimal mixing time was determined for the p9CREB peptide
sample
by comparing the NOE signal intensities in a series of experiments with mixing
times
10 varying from 100 msec to 750 msec. The chosen mixing time gave maximal NOE
buildup with no significant contribution from spin diffusion. This value was
used for
the non-phosphorylated analog experiment in order to maintain identical
experimental
conditions. Integrated peak volumes were converted into distance restraints
using a
r 6 dependency and the known distance of 2.47 ~ between the two adjacent
protons of
15 the tyrosine aromatic ring was used for calibration. The restraints were
classified into
strong (1.8-2.5 ~), medium (1.8-3.5 ~) and weak (1.8-5.0 ~). An empirical
correction of 0.5 ~r was added to the upper bound for restraints involving
methyl
groups.
The structures were calculated by hybrid distance geometry - dynamical
20 simulated annealing using XPLOR (version 3.856). The NOE energy was
introduced
as a square-well potential with a constant force constant of 50 Kcal/mol~t~2.
Simulated annealing consisted of 1500 3 fsec steps at 1000 K and 3000 lfsec
steps
during cooling to 300 K. Finally, the structures were minimized using
conjugate
gradient energy minimization for 4000 iterations. INSIGHTII (Molecular
Modeling
25 System version 97.0, Molecular Simulations, Inc.) was used for
visualization and
analysis of the NMR-derived structures. Their quality was assessed using
PROCHECK.
Analytical methods:
Proton, carbon, fluorine and phosphorus nuclear magnetic resonance spectra
30 were obtained on either a Broker AMX 500 spectrometer or a Brucker AV 300
spectrometer and are reported in ppm (8). Tetramethylsilane (TMS) was used as
an
internal standard for proton spectra, phosphoric acid was used as an internal
standard
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46
for phosphorus spectra and the solvent peak was used as the reference peak for
carbon
and fluorine spectra.
Mass spectra were obtained on a Finnigan LCQ Duo LC-MS ion trap
electrospray ionization (ESI) mass spectrometer.
Thin-layer chromatography (TLC) was performed using Analtech silica gel
plates and visualized by ultraviolet (CJV) light, or by staining the plates in
0.2 wt
ninhydrine in butanol.
Elemental analysis was performed by Quantitative Technologies, Inc.
(Whitehouse, NJ).
HPLC analyses were obtained using a Hypersil BDS C18 Column,
4.6 X 150 mm, 5 ~,m, Column Temperature Ambient, Detector @ 220 nm using a
standard solvent gradient program, as follows:
Time Flow %A %B
(Minutes) mL/min)
0.0 1.0 100 0.0
4.0 1.0 100 0.0
20.0 1.0 92.0 8.0
21.0 i.0 100 0.0
22.0 ~ 1.0 ~ 100 0 0
h - v. ~ io ~ r mn wafer
B = 0.1 % TFA in acetonitrile
In vitro inhibition assays:
Purified recombinant rabbit GSK-3~3 (Elder-Finkelman et al., 1995) was
incubated with peptide substrate PGS-1 (YRR.AAVPPSPSLSRHSSPSQS(p)EDEEE)
(SEQ ID NO:l) and with phenyl phosphate, pyridoxal phosphate (P-5-P), GS-1, GS-
2, GS-3, GS-5 or GS-21 (structural formulas are depicted in Figure 3), at
indicated
concentrations. The reaction mixture included Tris SO mM (pH = 7.3), 10 mM
MgAc,
saP[,~-ATP] (100 ~.M), 0.01 % ~3-mercaptoethanol, and was incubated for 10
minutes
at 30 °C. Reactions were spotted on phosphocellulose paper (p81),
washed with 100
mM phosphoric acid, and counted for radioactivity (as described in Elder-
Finkelman
et al., 1996).
Glucose uptake i~z isolated adipocytes: Mice adipocytes were isolated from
epididymal fat pad by digestion with 0.8 mglml collagenase (Worthington
Biochemical) as described previously (Lawrence et al., 1977). Digested fat
pads
were passed through nylon mesh and cells were washed 3 times with Krebs-
bicarbonate buffer (pH = 7.4) containing 1 % bovine serum albumin (Fraction V,
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47
Boehringer Mannheim, Germany), 10 mM HEPES (pH = 7.3), 5 mM glucose and 200
nM adenosine. Cells were incubated with GS-5 and GS-21 at indicated
concentrations for 2.5 hours, followed by addition of 2-deoxy [3H] glucose
(0.5
lCCi/vial) for 10 minutes. The assay was terminated by centrifugation of cells
through
dinonylphthalate (ICN, USA). 3H was thereafter quantitated by liquid
scintillation
analyzer (Packard). Nonspecific uptake of 2-deoxy-[3H] glucose was determined
by
the addition of cytochalasin B (50 lCM) 30 minutes prior to the addition of
radioactive
material.
to E~YPERIMENT~1L RESULTS
Determination of a 3D stYUCtu~e of a GSA 3 substrate:
Tables 2 and 3 below present the structural coordinate data that was used for
inputting into structure analysis software for visualization of the 3D
structures.
The obtained 3D structures, presented in Figures la and lb, it was observed
that only the phosphorylated peptide has a defined structural conformation. As
is
shown in Figure la, for p9CREB, the phosphorylation imposed a significant
"turn" of
the peptide backbone, bringing Tyr 8 and Arg 4 closer, and forming a 'loop
structure'
whereby the phosphorylated residue is pointing out of the loop. This
conformation
minimizes on the one hand interference of positively charged residues (Arg 4
and Arg
5) with the catalytic binding pocket of the enzyme, and on the other hand,
renders the
phosphorylated serine readily accessible to the enzyme. This structure
analysis
provides an explanation for the unique substrate recognition of GSK-3. The
design of
small molecules that mimic the structure presented here thus provides a method
for
obtaining potential selective inhibitors for GSK-3.
Chemical Syntheses:
Synthesis of p-methyl bend phosphate (GS 1):
The general synthesis of GS-1 is depicted in Scheme 1 below.
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48
Scheme 1
HO ~ t ~N.N ~ O_P OtBu O_P OH
1. Pr2N-P(O Buys, (V-~ O BU HCl/dioxane OH
_. ~ ~ 2. mCPBA~ ~ i
GS-1
Preparation of di-test=butyl, p-methyl ben~yl phosphate: 1H-Tetrazole
solution (0.45 M in acetonitrile, 20 ml, 9 mmol, 3 equivalents) was added in
one
portion to a stirred solution of 4-methylbenzyl alcohol (0.4 gram, 3.3 mmol,
1.1
equivalent) and di-tert-butyl diisopropyl phosphoramidite (0.95 ml, 0.83 gram,
3
mmol, 1 equivalent) in dry THF (3 ml). The mixture was stirred for 15 minutes
at 20
°C. The mixture was cooled to --40 °C (by means of dry
ice/acetonitrile), and a
solution of 85 % ~n-chloroperbenzoic acid (mCPBA) (0.81 gram in 1 ml
dichloromethane, 4 mmol, i .3 equivalents) in dichloromethan (4 mL) was
rapidly
added while the reaction temperature was kept below 0 °C. The solution
was allowed
to warm up to room temperature and after stirring for 5 minutes at 20
°C, 10
aqueous NaHSO3 (10 ml) was added and the mixture was stirred for a further 10
minutes. The mixture was then extracted with ether (70 ml) and the aqueous
phase
discarded. The ethereal phase was washed with 10 % aqueous NaHSO3 (2 x 20 ml),
5
saturated aqueous Na$COs (2 x 20 ml), dried on sodium sulfate and filtered.
The
organic filtrate was evaporated and the residue was purified by chromatography
on a
silica gel column, using a mixture of EtOAc/hexanes 1:15 as eluent, to give a
mixture
of the product (di-tert-butyl, p-methyl benzyl phosphate) and the starting
material,
which was used without further separation.
1H NMR (200 MHz, CDCl3): 8 = 7.22 (m, 4H, Ar), 4.93 (d, J = 7.22 Hz, 2H,
CH20), 2.33 (s, 3H, CH3), 1.46 (s, 18H, OtBu).
13C NMR {50.4 MHz, CDC13): b = 137.7 (Ar), 137.0 (Ar), 129.0 (Ar), 127.7
{Ar),82.3 (c), 68.3 (CH20), 29.8 {OtBu), 21.1 (CH3).
31P NMR (81.3 MHz, CDC13): 8 = -9.4 ppm.
Preparation of p-methyl ben~yl phosphate: A solution of HCl (4M in
dioxane, 2 ml, 8 mmol, 2.6 equivalents) and dioxane (6 ml) was added to the
obtained
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49
di-tert-butyl, p-methyl benzyl phosphate at 20 °C and the reaction was
monitored by
TLC. Once the hydrolysis was completed, the dioxane was evaporated under
reduced
pressure, and the residue was dissolved in water (15 ml) and washed with ether
(2 x
15 ml) to remove excess of the benzyl alcohol starting material. The solvent
was
then evaporated under reduced pressure and the resultant clear oil slowly
changed
into a colorless solid upon a prolonged high vacuum drying, to give 0.18 gram
(30 %)
of the final product.
1H NMR (200 MHz, D20): & = 7.27 (d, J = 8.1 Hz, 2H, Ar), 7.19 (d, J = 8.1
Hz, 2H, Ar), 4.82 (d, J = 7.0 Hz, 2H, CH20), 2.26 (s, 3H, CH3).
13C NMR (50.4 MHz, DSO): b = 138.7 (Ar), 134.0(Ar), 129.2 (Ar), 128.0 (Ar),
6s.o (cH2o), 20.1 (cH3).
31P NMR (81.3 MHz, DZO): 8 = 0.6 ppm.
Synthesis of ben~yl phosphate (GS 2):
The general synthesis of benzyl phosphate is depicted in Scheme 2
hereinbelow:
Scheme 2
HO ~ O_P O#Bu O-P OH
1.'P~ZN-P(OtBu~, N_N OtBU HCIfdioxane OH
2. mCPBA ~ i
2o GS-2
Preparation of di-tert butyl, benzyl phosphate: 1 H-Tetrazole solution (0.45 M
in acetonitrile 20 ml, 9 mmol, 3 equivalents) was added in one portion to a
stirred
solution of the benzyl alcohol (0.34 ml, 3.3 mmol, 1.1 equivalent) and di-tert-
butyl
diisopropyl phosphoramidite (0.95 ml, 0.83 gram, 3 mmol, 1 equivalent) in dry
THF
(3 ml). The mixture was stirred for 15 minutes at 20 °C and was
thereafter cooled to
-40 °C (by means of dry iceJacetonitrile). A solution of 85 % mCPBA
(0.81 gram in
1 ml dichloromethane, 4 mmol, 1.3 equivalents) in dichloromethane (DCM) (4 ml)
was rapidly added while the reaction temperature was kept below 0 °C.
The solution
was allowed to warm up to room temperature and after stirring for 5 minutes at
20 °C,
10 % aqueous NaHSOs (10 ml) was added and the mixture stirred for additional
10
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WO 2005/000192 PCT/IL2004/000570
minutes. The mixture was then extracted with ether (70 ml) and the aqueous
phase
discarded. The ethereal phase was washed with 10 % aqueous NaHS03 (2 x 20 ml),
5
saturated aqueous NaHC03 (2 x 20 ml), dried over sodium sulfate and filtered.
The organic layer was evaporated and the residue was purified by
chromatography on
5 a silica gel column using a mixture of EtOAc/hexanes 1:15 as eluent, to give
a
mixture of the product (di-tert-butyl, benzyl phosphate) and the starting
material,
which was used without fiu-ther purification.
1H NMR (200 MHz, CDCl3): 8 = 7.36 (m, SH, Ar), 4.99 (d, J = 7.33 Hz, 2H,
CH20), 1.46 (s, 18H, OtBu).
10 31P NMR (81.3 MHz, CDCl3): 8 = -9.3 ppm.
'reparation of bera~yl phosphate: A solution of HCl (4M in dioxane, 2 ml, 8
mmol, 2.6 equivalents) and dioxane (6 ml) was added to obtained di-tert-butyl,
benzyl
phosphate at 20 °C and the reaction was monitored by TLC. Once the
hydrolysis was
completed, the dioxane was evaporated under reduced pressure, and the residue
was
15 dissolved in water (15 ml) and washed with ether (2 x 15 ml) to remove
excess of the
benzyl alcohol starting material. The solvent was then evaporated under
reduced
pressure and the resultant clear oil slowly changed into a colorless solid
upon a
prolonged high vacuum drying, to give 0.17 gram (30 %) of the final product.
1H NMR (200 MHz, D20): 8 = 7.34 (m, SH, Ar), 4.82 (d, J = 7.09 Hz, 2H,
20 CH20).
31P NMR (81.3 MHz, D20): ~ = 0.7 ppm.
Synthesis of 3 pyridylmethyl phosphate (GS 3):
The general synthesis of 3-pyridylmethyl phosphate is depicted in Scheme 3
below:
Scheme 3
HO ~ O_~ OtBu O_P OH
' 1. ~Pr2N-P{OtBu~, N_N OtBU HClldi~xane OH
1 '
'N 2.H2o2 ~ .N ~ ,N
GS 3
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51
Prepa~atio~a of di-test butyl, 3-Pyridylmethyl phosphate: 1H-Tetrazole
solution (0.45 M in acetonitrile, 20 ml, 9 mmol, 3 equivalents) was added in
one
portion to a stirred solution of 3-pyridylmethanol (0.31 ml, 3.3 mmol, 1.1
equivalent)
and di-tert-butyl diisopropyl phosphoramidite (0.95 ml, 0.83 gram, 3 mmol, 1
equivalent) in dry THF (3 ml). The mixture was stirred for 15 minutes at 20
°C and
was thereafter cooled to -40 °C (by means of dry ice/acetonitrile). A
solution of 85
mCPBA (0.81 gram in 1 ml DCM, 4 mmol, 1.3 equivalents) in DCM (4 ml) was then
rapidly added while the reaction temperature was kept below 0 °C. The
solution was
allowed to warm up to room temperature and after stirring for 5 minutes at 20
°C,
10% aqueous NaHS03 (10 ml) was added and the mixture was stirred for
additional
10 minutes. The mixture was then extracted with ether (70 ml) and the aqueous
phase
discarded. The ethereal phase was washed with 10 % aqueous NaHS03 {2 x 20 ml),
5
saturated aqueous NaHC03 (2 x 20 ml), dried over sodium sulfate and filtered.
The organic filtrate was evaporated and the residue was purified by
chromatography
on a silica gel column using a mixture of CHC13/hexanes l:l as eluent, to give
a
mixture of di-tert-butyl, 3-Pyridylmethyl phosphate and the starting material,
which
was used without further purification.
1H NMR {200 MHz, CDC13): b = 8.52 (d, J = 1.56 Hz, 1H, Ar), 8.51 {dd, J =
4.83 Hz, J = 1.53 Hz, 1H, Ar), 7.80 {m, 1H, Ar), 7.33 (dd, J = 7.44 Hz, J =
4.62 Hz,
1H, Ar), 4.94 (d, J = 6.83 Hz, 2H, CHaO), 1.46 (s, 18H, OtBu).
isC NMR {50.4 MHz, CDCl3): b = 148.2 (Ar), 148.1 (Ar), 135.3 (Ar), 134.3
{Ar), 123.1 (Ar),77.9 (c), 64.1 (CH20), 29.6 (OtBu).
31P NMR {81.3 MHz, CDC13): S = -9.4 ppm.
P~eparatior~ of 3 pyridylmethyl phosphate: A solution of HCl (4M in
dioxane, 2 ml, 8 mmol, 2.6 equivalents) and dioxane (6 ml) was added to the
obtained
di-tert-butyl, 3-Pyridylmethyl phosphate at 20 °C and the reaction was
monitored by
TLC. Once the hydrolysis was completed, the dioxane was evaporated under
reduced
pressure, and the residue was dissolved in water (15 ml) and washed with ether
(2 x
15 m1). The solvent was then evaporated under reduced pressure to give 0.19
gram
(30 %) of the final product.
1H NMR (400 MHz, D20): 8 = 8.72 (t, J = 0.81 Hz, 1H, Ar), 8.62 (d, J = 5.71
Hz, 1 H, Ar), 8.51 (d, J = 8.27 Hz, 1 H, Ar), 7.96 (dd, J = 8.1 S Hz, J = 5.94
Hz, Ar),
5.04 (d, J = 8.23 Hz, 2H, CH20).
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52
i3C NMR {100.8 MHz, D20): b = 144.9 (Ar), 139.9 (Ar), 139.0 (Ar), 126.8
(Ar), 62.8 {CH20)-.
3 iP NMR (162 MHz, D20): b = 0.76 ppm.
Synthesis of 3,5-bis(2-aminoethyl)ben~yl phosphate (GS 21):
A general strategy for synthesizing GS-21, depicted in Scheme 4 below, as
well as a purification protocol of the final product, were designed and
practiced. 3,5-
Bis(2-aminoethyl)benzyl phosphate (GS-21) was obtained in 5 % overall yield by
an
eight-step synthesis. The corresponding trifluoroacetic acid salt was also
prepared.
l0 Scheme 4
COZMe OH O.P~OH
n
~ O
------
MeOZC I / COZMe HzN I / NHz H N I / NH
z z
GS-21
The benzyl alcohol intermediate (see, Scheme 4) was identified as a key
intermediate obtainable in four steps from the inexpensive starting material
trimethyl
1,3,5-benzenetricarboxylate, as is detailed hereinbelow and is depicted in
Scheme S.
Scheme S
COZMe OH OH
LAH, THF TMSBr, CH3CN
94% ~ 57%
I HO I / OH Br I / Br
MeOZC / COzMe
NaCN
MeOH/Hz0
89%
OH OH
Raney Ni, HZ
quantitative N \
/ ~ I / ~N
HzN NHS
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53
The phosphate moiety was then introduced by reaction of the alcohol with di-
tert-butyl diisopropyl phosphoramidite, in the presence of tetrazole,
according to the
method of Johns (Tetrahedron Lett. 1988, 29, 2369-2372). Immediate oxidation
without isolation of the resulting phosphite by m-chloroperbenzoic acid
{mCPBA)
yielded the corresponding phosphate ester. Global deprotection of the amines
and the
phosphate was achieved by the use of trifluoroacetic acid under controlled
conditions.
The material was then obtained as its trifluoroacetate salt. The latter was
recrystallized prior to treatment with an ion-exchange resin to afford the
desired
product with adequate purity typically approximately 90 % (AUC by HPLC), as
l0 depicted in Scheme 6 below.
Scheme 6
OH O' OOH
P-OH
~i
' \ \ 0
HzN NH2 H2N / NHZ
GS-21
BoczO 1) TFA
70% 2) Dowex 50
i-Pr 26%
,N, ~Ot-Bu Ot Bu
OH i-Pr P O, P~Ot-Bu m-CPBA O,
P-Ot Bu
Ot Bu i 61%, 2 steps
\ tetrazole I \ Ot-Bu ~ I \ O
BocHN NHBoc BocHN NHBoc BocHN / NHBoc
IS Following is a detailed description of the synthesis:
Preparation of 1,3,5 Tris(hydroxylmethyl)benzene: A 3-liter, round-bottom
flask equipped with an overhead stirrer, an addition funnel and a reflux
condenser was
charged with lithium aluminum hydride {49.7 grams, 1.31 mol) and anhydrous THF
(500 ml) under nitrogen atmosphere. The resulting suspension was slowly heated
to
20 reflux and a solution of trimethyl-1,3,5-benzenetricarboxylate (100.0
grams, 0.40
mol) in anhydrous THF (1.0 liter) was added dropwise thereto while maintaining
a
. gentle reflux (3 hours). The resulting gray suspension was stirred under
reflux for
additional 7 hours and then cooled in an external ice-water bath. The excess
lithium
aluminum hydride was hydrolyzed by dropwise addition of water (50 ml, 45.
minutes),
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S4
then 1 S % NaOH (S 0 ml, slow stream), and finally more water ( 1 S 0 ml, slow
stream).
The resulting suspension was stirred at ambient temperature for 14 hours. The
solids
were filtered off and the filtrate was concentrated under high ,vacuum to
obtain a
colorless oil which slowly solidified to afford 1,3,5-
tris(hydroxylmethyl)benzene
{62.3 grams, 94 %) as a white solid. The'H NMR and i3.C NMR spectra, presented
in
Figures 4a-b, were consistent with the assigned structure.
PrepaYatioh of 3,5-Bis(bromomethyl)benzyl Alcohol: A 2-liter, round-bottom
flask equipped with a magnetic stir bar and an addition funnel was charged
with 1,3,5-
tris(hydroxylmethyl)benzene [33.7 grams, 0.20 mol) and anhydrous acetonitrile
(7S0
ml). To the resulting suspension was added, with stirring,
bromotrimethylsilane
(TMSBr) 979.0 ml, 0.60 mol) as a slow stream. The white slurry turned brown
and
viscous. The reaction mixture was then heated to 40 °C for 2S minutes
and resulted in
a clear solution. The reaction was judged to be complete by TLC analysis
(90:10
methylene. chloride/methanol, visualization by UV, starting material Rf 0.07,
product
Rf 0.77). The solvent was removed under reduced pressure to obtain a brown
paste.
The crude material was purified by column chromatography (silica gel, 0-S
MeOH/CHZC12). 3,S-Bis(bromomethyl)benzyl alcohol {33.5 grams, S7 %) was
obtained as a white solid. The 1H NMR and '3C NMR spectra, presented in Figure
Sa-b, were consistent with the assigned structure.
Preparation of 3,S Bis(cyanomethyl)benzyl Alcohol: A 1-liter, round-bottom
flask equipped with an overhead stirrer, an addition funnel and a ref(ux
condenser was
charged with 3,S-Bis(bromomethyl)benzyl alcohol (33.1 grams, 0.11 mol) and
methanol (400 ml). The resulting clear solution was heated to reflex. A
solution of
sodium cyanide {16.2 grams, 0.33 mol) in water (2S ml) was added slowly.
Heating
was continued under reflex for 6 hours before the reaction was judged to be
complete
by TLC analysis {9S:S methylene chloride/methanol, visualization by UV,
starting
material Rf 0.62, product Rf 0.38). The reaction mixture was cooled to ambient
temperature and solvent was removed under reduced pressure to obtain a brown
paste.
The latter was triturated with MTBE (6 ~ 100 ml). The MTBE extracts were
combined and the solvent removed under reduced pressure. The yellow oil thus
obtained was then purified by column chromatography (silica gel, 0-S
MeOH/CH2C12). 3,S-Bis(cyanomethyl)benzyl alcohol (18.7 grams, 89 %) was
obtained as a light brown oil, which slowly tuxned into a waxy white solid. A
1-gram
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sample was removed and purified via column chromatography to give a purified
sample. The iH NMR and 13C NMR spectra, presented in Figures 6a-b, were
consistent with the assigned structure.
Preparation of 3,5 Bis(amir~oethyl)berazyl Alcohol: A sample of 3,5
5 Bis(cyanomethyl)benzyl alcohol (8.0 grams, 0.04 mol) was divided in three
parts and
each 2:5- to 3.0-grams portion was charged into separate 500-ml Parr bottles,
followed by ethanol (100 ml), and aqueous NaOH (1.2 grams in 5 ml of water).
To
the resulting solution was added Raney Ni (50 % suspension in water, 1.2
grams).
The mixture was hydrogenated at 30 psi on a Parr shaker. The reaction was
l0 monitored by 1H N1VIR and judged complete after 3 hours. The catalyst was
filtered
on a pad of diatomaceous earth and the diatomaceous earth pads washed with
ethanol
(200 ml). The filtrates from all three reactions were combined and solvent
removed
under reduced pressure to obtain 3,5-bis(aminoethyl)benzyl alcohol as a brown
paste
of {14.16 grams). 1H NMR spectrum of the product, presented in Figure 7, shows
15 presence of 25 % (w/w) of ethanol. No noticeable change in ethanol content
was
observed when the sample was dried under high vacuum for an extended period of
time. This material was used in the next step of the synthesis without any
further
purification.
Preparation of 3,5-Bis(tert butoxyca~bonylamir~oethyl)ber~zyl Alcohol: A
20 three-neck, 3-liter, round-bottom flask equipped with a magnetic stir bar,
thermometer
and gas inlet adapter was charged with 3,5-bis(aminoethyl)-1-
hydroxymethylbenzyl
alcohol (29.4 grams) dissolved in THF {590 ml) and 2 N aqueous NaOH (590 ml).
To
the stirred mixture was added di-tert-butyl dicarbonate {59.4 grams, 272 mmol)
in one
portion. The mixture was heated to 45 °C for 4 hours. The resulting
solution was
25 cooled to ambient temperature and the volatile organics were removed by
vacuum.
To the resulting water mixture was added methanol (600 ml). The stirred
solution
was heated to 45 °C for 2 days in order to selectively hydrolyze the
tent-
. butoxycarbonate moiety while preserving the carbamates. After cooling to
ambient
temperature the solution had volatiles removed and the aqueous mixture was
extracted
30 with chloroform (3 ~ 600 ml). The organic layers were combined, washed with
brine
(600 m1) and concentrated. After drying under high vacuum, crude 3,5-bis(te~t-
butoxycarbonylaminoethyl)benzyl alcohol (24.88 grasps) was obtained in 67 %
yield
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56
as an off white solid. The 1H NMR spectrum, presented in Figure 8, was
consistent
with the assigned structure. The crude material was used without further
purification.
Preparation of protected 3,S-Bis(2-aminoethyl)benzyl Phosphate: A 500-ml,
round-bottom flask equipped with a magnetic stir bar and an addition funnel
was
charged with 3,5-bis(test-butoxycarbonylaminoethyl)benzyl alcohol (2.3 grams,
0.0058 mot) and anhydrous methylene chloride (45 ml). The resulting solution
was
cooled to approximately 5 °C in an ice-water bath. A solution of di-
tert-butyl
diisopropylphosphoramidite (4.5 ml, 0.0144 mol) in anhydrous acetonitrile (45
ml)
was added as a slow stream from the addition funnel. A solution of tetrazole
(1.0
grams, 0.0144 mol) in a 1:1 mixture of anhydrous acetonitrile/anhydrous
methylene
chloride (90 ml) was then added slowly (15 minutes). The resulting white
suspension
was stirred at approximately 5 °C for 1 hour and the reaction was
judged complete by
TLC analysis (95:5 chloroform/isopropyl alcohol, visualization by staining in
ninhydrin, starting material Rf0.23, product Rf0.30). The solvent was removed
under
reduced pressure to obtain a paste, which was dissolved in anhydrous methylene
chloride (75 ml) and cooled in a dry ice/acetonitrile bath. A solution of
mCPBA (1.3
grams, 0.0144 mol) in anhydrous methylene chloride (50 ml) was added all at
once.
The resulting mixture was stirred for 1 hour, allowed to warm up to ambient
temperature (1 hour) and stirred for another 30 minutes. The reaction mixture
then
was washed successively with 1.0 M aqueous solution of sodium thiosulfate (100
ml)
and saturated sodium bicarbonate (2 ~ 100 ml). The organic extract was dried
over
anhydrous sodium sulfate and filtered. The solvent was removed under reduced
pressure to obtain the crude phosphate as a yellow oil, which was then
purified by
column chromatography (silica gel, 0-5 % MeOH/CH2C12). The protected 3,5-Bis(2-
aminoethyl)benzyl phosphate (2.1 grams, 61 %) was obtained as a viscous,
colorless
oil. The 1H NMR, 13C NMR and Sip IVMR spectra thereof, presented in Figures 9a-
c,
were consistent with the assigned structure.
Preparation of 3,S Bis(2-aminoethyd)benzyl Phosphate TFA Salt: A 250-ml,
round-bottom flask. was equipped with a magnetic stir bar was charged with the
protected phosphate (2.9 grams, 0.0049 mol), anhydrous dichloromethane (30 ml)
and
trifluoroacetic acid (30 ml). The resulting clear solution was stirred at
ambient
temperature for 3 hours. The reaction was judged complete by 1H NMR and 31P
NMR analysis. Removal of the solvent under reduced pressure afforded a viscous
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57
orange oil, which . was dissolved in methanol (7.5 ml) and added with stirring
to
diethyl ether (S00 ml), resulting in precipitation of the product. The
resulting slurry
was stirred for 1 hour at ambient temperature and then solids allowed to
settle. The
clear solution was decanted off from the top and the product triturated with
ether (2 ~
100 ml). Each time the solids were allowed to settle and the clear solution
was
decanted off. The product was finally dried in a vacuum oven for 108 hours at
55 °C
and then for an additional 192 hours at 65 °C to afford 3,5-bis(2-
aminoethyl)benzyl
phosphate TFA salt (1.78 grams, 71 %) as a white solid. The'H NMR, 13C NMR and
3iP NMR spectra, presented in Figures 10a-c were consistent with the assigned
structure. The 1H NMR spectrum showed the presence of 8.5 % (w/w) of ether in
the
product. This ether proved very difficult to remove and the material was
characterized as hygroscopic. Mass spectrum of the product, presented in
Figure l Od,
indicated a molecular peak at mlz 275 [C11H19N204P + H]+.
Preparation of 3,5-Bis(2-aminoethyd)behzyl Phosphate (GS 2~): A three
neck, 5-liter, round-bottom flask equipped with an overhead mechanical
stirrer,
thermometer, 1-liter pressure-equalizing addition funnel and gas inlet adapter
was
charged with a solution of 3,5-bis(tart-butoxycarbonylaminoethyl)benzyl
alcohol
(24.88 grams, 63.15 mmol) in anhydrous dichloromethane {1 1) under nitrogen
atmosphere. The reaction mixture was cooled with an ice/brine bath. Di-tart-
butyl
diisopropylphosphoramidite (49.8 ml, 157.9 mmol) in anhydrous acetonitrile (1
liter)
was added via the pressure-equalizing addition funnel at such a rate that the
reaction
temperature was maintained <6 °C. Tetrazole (351 ml of a 0.45 M
solution in
acetonitrile, 157.9 mmol) was diluted with anhydrous acetonitrile (150 ml) and
anhydrous dichloromethane {500 ml) and added via the pressure-equalizing
addition
funnel at such a rate that the temperature was maintained under 6 °C.
After the
addition was completed, the flask was left in the cold bath and the reaction
mixture
stirred for 1 hour. The flask was then cooled to -35 °C by means of dry
ice/acetonitrile bath. A solution of 3-chloroperoxy-benzoic acid ( 18.4 grams,
82.1
mmol) in anhydrous dichloromethane (500 ml) was added in one portion. The
mixture was allowed to warm to ambient temperature and thereafter stirred for
2
hours. The solution was poured into a solution of Na2S20s {20 grams). and
KZCO3 (50
grams) in water (1.5 liters). The resulting biphasic mixture had a pH of 11.
After
stirring for 15 minutes the volatile organics were removed in vacuum and the
water
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5~
layer was extracted with chloroform (4 x 750 ml). The combined organic layers
were
dried over magnesium sulfate, filtered and concentrated to a yellow oil (69
grams).
The crude material was purified by column chromatography (silica gel,
MTBE/heptane, 6:4). Mixed fractions containing the product were combined and
concentrated to a light yellow oil (32.6 grams). A 28.6-grams portion of the
oil was
dissolved into dichloromethane (287 ml, 10 volumes) and charged into a 1-
liter,
round-bottom flask equipped with a 500-ml pressure-equalizing addition- fLwnel
and
magnetic stir bar. Trifluoroacetic acid (287 ml, 10 volumes) was added rapidly
via
the pressure-equalizing addition funnel. The resulting solution was stirred
for 5
hours. After concentrating and drying overnight under high vacuum, a thick
orange
oil (37.88 grams) was obtained. The residue was dissolved in water (57 ml, 1.5
volumes) and added dropwise into stirred methanol {90 volumes) yielding a
precipitate. After stirring for 30 minutes, the solids were allowed to settle
for 1 hour
and the liquid was decanted off. The remaining liquid was removed in vacuum
giving
13.72 grams of solid. The material was dissolved in water (68 ml, 5 volumes)
and
loaded onto Dowex SOWXB-200 ion-exchange resin (137 grams). The column was
washed with water (550 ml, 40 volumes). The product was eluted with 3:1
MeOH/aqueous NH40H (2 liters, 145 volumes). The methanol fractions were
concentrated under reduced pressure to yield an off white solid. The solid was
dissolved in a minimum amount of water and added into stirred methanol (40
volumes). The precipitate was collected via filtration and dried overnight
under
vacuum. The resulting powder was triturated with water (7 volumes). After
filtration
and drying under high vacuum, the final product (2.0 grams) was obtained as a
white
powder. The filtrate was concentrated and the residue triturated with water (5
volumes). After filtration and drying under high vacuum, a second crop of
product
[0.9 grams) was obtained. The two lots were combined and blended for 10
minutes.
3,5-Bis(2-aminoethyl)benzyl phosphate (GS-21) was obtained as a white powder.
The 1H NMR, 31P NMR, and 13C NMR spectra of the product, presented in Figures
11 a-c, were consistent with the assigned structure. The MS spectrum,
presented in
Figure lld, indicated a molecular peak at 275 [C11H19Nz04P + H]+. HPLC
chromatogram (obtained using method A described above), presented in Figure
11e,
showed a 96.7 % purity of the product. The final product was characterized as
non-
hygroscopic.
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59
Using the same strategy, the novel compounds GS-4 and GS-5 were
synthesized as follows:
Synthesis of 3-(guanidi~tomethy) be~azyl phosphate (GS 4):
The general synthesis of GS-4, as its trifluoroacetic acid salt, is depicted
in
Scheme 7 below:
Scheme 7
OH
HO H~ N N'-bis(tert-butoxycarbonyl)
LiAIH4 -S-methylisothiourea I ~ 1.'Pr2N-P(O~Bu)Z, N-nj
~NH
H N i Et N 2. mCPBA
N z ~ a
HzN NHz
O_ P OH
'Bu
O B O_
TFAIDCM OH
~
NH \
~NH
Hz~ NHz ~
NHz
Hzt~
cF3coz
GS-4 (TFA salt)
Preparation of 3-(aminomethyl)beu~yl alcohol: A solution of 3-
(hydroxymethyl)benzonitrile in THF was slowly added to a refluxing solution of
LiAlH4 in THF with vigorous stirring, maintained under nitrogen atmosphere.
The
solution was heated at reflux overnight and water was thereafter slowly added
dropwise to quench the reaction (until no fiu~ther evolution of HZ was
apparent). The
THF was evaporated under reduced pressure and etherlacidified water was added.
The
ether phase was discarded. The aqueous phase was washed with ether and the
organic
phase was discarded. NaOH was added until the pH of the aqueous phase reached
pH
7. The solution was extracted with THF three times, dried over MgS04 and
evaporated under reduced pressure to fwnish a slightly yellow residue which
was
purified by chromatography on a silica gel column using a gradient eluent
starting
from ethyl acetate and ending with a mixture of 1:1 ethyl acetate:MeOH), to
give the
intermediate in a 40 % yield.
1H NMR (200 MHz, d6-DMSO): b = 7.16-7.27 (m, 4H, Ar), 4.47 (s, 2H,
CHZO), 3.94 {bs, 2H, NH2), 3.72 (s, 2H, CH2NH2).
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13C NMR (50.4 MHz, CDC13) 8 = 143.1, 142.8, 128. 2, 125.9, 125.7, 124.9,
63.3, 45.6.
Pweparation of 3-(N,N'-bis-BOC guanidir~omethyl) ben~yl alcohol: A
solution of 3-(aminomethyl)benzylalcohol, 3-(N,N'-bis(tert-butoxycarbonyl)-S-
methylisothiourea and triethyl amine in dry DMF was stirred at room
temperature
overnight. An ether/water mixture was then added and the organic layer
separated,
while the aqueous layer was extracted with ether. The combined organic extract
was
washed with water, dried over MgS04 and evaporated under reduced pressure. The
crude product was purified by flash chromatography using a gradient eluent
starting
from hexanes and ending with a mixture of 1:5 ethyl acetate: hexanes), in 85 %
yield.
1H NMR (200MHz, CDC13): ~ = 11.51 (bs, 1H), 8.56 (bs, 1H), 7.22-7.38 (m,
4H), 4.70 (s, 2H), 4.65 (d, J--S.lHz, 2H), 1.52 (s, 9H), 1.48 (s, 9H).
13C NMR (50 MHz, CDCl3): ~ = 155.9, 153.1, 141.5, 137.7, 128.9, 127.0,
126.4, 126.2, 65.0, 45.0, 28.2, 27.9.
Preparation of Di-tert butyl, 3-(N,N'-bis-BOC=guanidihomethy) ben~yl
phosphate: 1H-Tetrazole solution (0.45 M in acetonitrile, 20 ml, 9 mmol, 3
equivalents) was added in one portion to a stirred solution of 3-(N, N-bis-BOC-
guanidinomethyl)benzyl alcohol (1 equivalent) and di-tert-butyl diisopropyl
phosphoramidite ( 1.42 ml, 1.24 grams, 4.5 mmol, 1.5 equivalents) in dry THF
(3 ml).
The mixture was stirred for 30 minutes at 20 °C and was then cooled to -
-40 °C (by
means of dry icelacetonitrile). A solution of 85 % mCPBA (1.25 grams in 1.5 ml
DCM, 6.15 mmol, 2.0 equivalents) in DCM (4 ml) was rapidly added while keeping
the reaction temperature below 0 °C. The solution was allowed to warm
up to room
temperature and, after stirring for 20 minutes, 10 % aqueous NaHS03 (10 ml)
was
added and the mixtuxe was stirred for additional 5 minutes. The mixture was
extracted with ether (70 ml) and the aqueous phase discarded. The ethereal
phase was
washed with 10 % aqueous NaHS03 (2 x 20 ml) and saturated aqueous NaHC03 (2 x
20 ml), dried over sodium sulfate and filtered. The organic filtrate was
evaporated
and the residue was purified by chromatography on a silica gel column using a
gradient eluent of ethyl acetate/hexanes 1:9 to 1:5), to give a mixture of the
phosphate ester product and the benzyl alcohol starting material, which was
further
purified by chromatography on a silica gel column., using a gradient eluent of
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61
CHCI3:MeOH 30:1 to 20:1), to give pure di-tert-butyl, 3-(N,N'-bis-BOC-
guanidinomethy) benzyl phosphate in 70 % yield.
1H NMR {200 MHz, CDC13): 8 = 11.52 (bs, 1H), 8.53 (bs, 1H), 7.25-7.35 (m,
4H), 4.99 (d, J=7.2 Hz, 2H), 4.63 (d, J--5.lHz, 2H), 1.51 (s, 9H), 1.47 {s,
27).
31P NMR (162 MHz, CDC13): 8 = -9.3.
Preparation of 3-(guanidinomethy) ben~yl phosphate, trifluo~oacetic acid
salt: A solution of 25 % trifluoroacetic acid (TFA) in DCM was added to di-
tert-
butyl, 3-(N,N-bis-BOC-guanidinomethy) benzyl phosphate at 20 °C and the
reaction
mixture was stirred for 18 hours. The solvent and TFA were thereafter
evaporated
under reduced pressure, and the residue was dissolved in water and washed with
ether. The solvent was then evaporated under reduced pressure, to give the
pure
product in 60 % yield.
1H NMR (200 MHz, D20): 8 = 7.25-7.35 (m, 4H), 4.84 (d, J=7.2Hz, 2H), 4.37
{s, 1H).
31P NMR (162 MHz, CDC13): 8 = 0.85.
19F~:8=-76.6.
Synthesis of 3 guanidinobenzyl phosphate (GS-S):
The general synthesis of GS-5, as its trifluoroacetic acid salt, is depicted
in
Scheme 8 below:
Scheme 8
N,N'-bis(tert-butoxycarbonyl~ N-N
\ S-methylisothiourea I \ t. iPrZN-P(°'Buh NN~ I \ ~ \
~ TFA~DCM
HZN~ HgClz ~ ~ z.m--crsn~ ~ ~ ~ HN~ O
Et3N ~ ~ °O v of
OH C OH C OP° C~ + OPw
HN~ ~NBOC HN~ ~NBOC IOBOtBu FizN \NHz \OH
BOC BOC
CF3C02
GS-5 TFA salt
Preparation of 3-(N,N! bis-BOC guanidino)ben~yl alcohol: A solution of
N,N'-bis(tent-butoxycarbonyl)-S-methylisothiourea (1.32 gram, 4.4 mmol, 1.1
equivalents), mercury chloride ( 1.22 gram, 4.4 mmol, 1.1 equivalents) and
triethylamine (1.72 ml, 12 mmol, 3 equivalents) was added to 3-aminobenzyl
alcohol
(0.5 gram, 4 mmol, 1.0 equivalent) in dry dimethylformamide (DMF) arid the
reaction
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62
mixture was stirred at room temperature for 5 hours. The mixture was
thereafter
extracted with ether/water and the organic layer was washed with saturated
aqueous
NH4C1 and brine. The aqueous layer was extracted with ether. The combined
ether
solution was dried over MgS04 and evaporated under reduced pressure. The crude
product was purified by flash chromatography on silica gel using a gradient
eluent of
hexanes to 40:60 ethyl acetate:hexanes), to give the intermediate in 60 %
yield.
1H NMR {200 MHz, CDC13): 8 = 11.60 (brs, 1H), 10.30 (brs, 1H), 7.11-7.55
(m, 4H), 4.65 (s, 2H), 1.49 (s, 9H), 1.48 (s, 9H).
13C NMR (400 MHz, CDC13): b = 171.4, 163.4, 153.7, 142.0, 136.7, 129.0,
123.4, 121.4, 120.8, 65.8, 64.7, 28.1, 27.9.
Preparation of di-tart butyl, 3-(N,N'-bis-BOC guanidino)ben~yl phosphate:
1-H-tetrazole solution (0.45 M in acetonitrile, 18.4 ml, 8.3 minol, 3
equivalents) was
added in one portion to a stirred solution of 3-(N,N'-bis-BOC guanidino)benzyl
alcohol (1 gram, 2.8 mmol, 1 equivalent) and di-tart-butyl diisopropyl
phosphoramidite (1.13 ml, 3.6 mmol, 1.3 equivalents) in dry THF (3 ml). The
mixture was stirred for 30 minutes at 20 °C and thereafter cooled to --
40 °C (by means
of dry ice/acetonitrile). A solution of 85 % mCPBA {0.85 gram in 1.5 ml DCM,
4.20
mmol, 1.5 equivalents) in DCM (4 ml) was rapidly added while keeping the
reaction
temperature below 0 °C. The reaction was allowed to reach room
temperature and
after stirring for 20 minutes, 10 % aqueous NaHSO3 (10 ml) was added and the
mixture stirred for additional 10 minutes. The mixture was extracted with
ether (50
ml) and the aqueous phase discarded. The ethereal phase was washed with 10
aqueous NaHS03 (2 x 20 ml) and saturated aqueous NaHC03 {2 x. 20 ml), dried
over
MgS04 and filtered. The solvent was evaporated and the residue was purified by
chromatography on a silica gel column using a gradient eluent of ethyl
acetate/hexanes 10:90 to 30:70), to give the protected product in 60 % yield.
1H NMR {200 MHz, CDC13): 8 = 11.65 (brs, 1H), 10.40 (brs, 1H), 7.10-7.64
(m, 4H), 4.97 (d, J=7.OHz, 2H), 1.49 (s, 18H), 1.45 (s, 18H).
13C NMR (400 MHz, CDC13): 8 = 153.3, 136.6, 129.2, 124.3, 122.1, 121.3,
67.9, b 29.8, 28Ø
mp NMR(200 MHz, CDC13): 8 = -9.3.
Preparation of 3 guanidinobcn~yl phosphate, trifluoroacetic acid salt: A
solution of 25 % TFA (1.5 ml) in DCM (4.5 ml) was added to di-tart-butyl-3-
(N,N'-
CA 02530111 2005-12-20
WO 2005/000192 PCT/IL2004/000570
63
bis-BOC guanidino)benzyl phosphate (0.3 gram, 0.54 mmol, 1 equivalent) at 20
°C
and the reaction mixture was stirred for 18 hours. The solvent and TFA were
thereafter evaporated under reduce pressure and the residue was dissolved in
water
and washed with ether. The solvent was evaporated under reduced pressure
(lyophilizerj, to give the pure product in 40 % yield (C1~H13F3N306P; Mw =
359.2
gramslmol).
1H NMR (200 MHz, CDCl3): 8 = 7.13-7.38 (m, 4H), 4.83 (d, J=7.6Hz, 2H)
13C NMR (400 MHz, CDCl3): 8 = 156.3, 139.5, 134.3, 130.1, 126.8, 125.3,
124.6, 66.4.
IO 31P NMR (200 MHz, CDC13): 8 = 0.8.
In vitro inhibition assays.
In a preliminary inhibition assay, the GSK-3 inhibition activity of the known
compounds phenyl phosphate, pyridoxal phosphate, GS-1, GS-2 and GS-3 was
tested
as described hereinabove. The results, presented in Figure 12 indicate that
all the
tested compounds exerted an inhibition activity toward GSK-3, with the
phosphate
derivatives of pyridine, namely, pyridoxal phosphate and GS-3, being more
active
than the phosphate derivatives of phenyl (phenyl phosphate, GS-1 and GS-2).
In an additional inhibition assay, the GSK-3 inhibition activity of GS-l, GS-
2.
GS-3, GS-5 and GS-21 was tested. The ability of GSK-3 to phosphorylate PGS-1
peptide substrate was measured in the presence of indicated concentrations of
these
compounds. The results, presented in Figure 13, represent the percentage of
GSK-3
activity as compared with a control incubation without inhibitors and are mean
of 2
independent experiments ~ SEM, where each point was assayed in triplicate.
As is shown in Figure 13, all the tested compounds were found highly active
in inhibiting GSK-3 activity (IC50 values of 1-5 mlV1], with GS-3 and GS-S
being the
most active compounds. These results may suggest that the presence of one or
more
nitrogen atoms in the ring or at an adjacent position thereto (e.g., directly
attached to a
ring atom) is a feature that may affect (enhance) the GSK-3 inhibition
activity of
newly designed small molecules.
Glucose Uptake:
The ability of the newly designed compounds GS-S and GS-21 to promote
glucose uptake was tested in mouse primary adipocytes as described
hereinabove.
The relative [3HJ 2-deoxy glucose incorporation observed in non-treated
adipocytes
CA 02530111 2005-12-20
WO 2005/000192 PCT/IL2004/000570
64
was normalized to 1 unit and the values obtained for [3H] 2-deoxy glucose in
adipocytes treated with GS-5 or GS-21 are presented as fold activation over
cells
treated with the peptide control, and are the mean of 6 independent
experiments ~
SEM, where each point was assayed in triplicate.
The results, presented in Figures 14a (GS-5) and Figure 14b (GS-21) show
that GS-21, at concentrations of 5 ~,M and 0.5 plVl increased glucose uptake
by 2.5-
fold and 1.7 fold, respectively. A somewhat reduced effect was observed in the
presence of GS-5, which enhanced glucose uptake approximately by 2-fold at a
concentration of 10 ~.M. As is further shown in Figures 14a and 14b, the
activation of
glucose uptake achieved by GS-5 and GS-21 was comparable to that achieved in
the
presence of 100 nM insulin. These results further demonstrate the ahilitv of
these
newly designed compounds to act as insulin mimetics in potentiating insulin
signaling
and treating GSK 3 mediated disorders such as diabetes.
CA 02530111 2005-12-20
WO 2005/000192 PCT/IL2004/000570
Table 2
REMARK FILENAME=~~refine 1 4.pdb~~
REMARK
REMARKoverall,bonds,angles,improper,vdw,noe,cdih
REMARKenergies: 49.6206, 2.76302, 89, 18, 24.9424,
18.0 2.93 0.894357,
$CDIH
REMARK
REMARKbonds, ers,noe,cdih
angles,improp
REMARKrms-d: -03,0.622994,0.465061,9.195132E-02,0
4.019702E
REMARK
REMARKnoe,cdih
REMARKviolations.: 0
0,
REMARK
REMARKDATE:27-Apr-00 08:12:42 c reated orish
by
user:
ATOM 1 CA ILE 1 11.861 -0.265 -1.7551.00 0.00
ATOM 2 HA ILE 1 11.773 0.710 -1.3051.00 0.00
ATOM 3 CB ILE 1 11.788 -0.143 -3.2781.00 0.00
ATOM 4 HB ILE 1 10.810 0.216 -3.5641.00 0.00
ATOM 5 CG1 ILE 1 12.034 -1.514 -3:9111.00 0.00
ATOM 6 HG11 ILE 1 12.789 -2.041 -3.3471.00 0.00
ATOM 7 HG12 ILE 1 12.368 -1.385 -4.9301.00 0.00
ATOM 8 CG2 ILE 1 12.852 0.841 -3.7661.00 0.00
ATOM 9 HG21 ILE 1 13.794 0.326 -3.8801.00 0.00
ATOM 10 HG22 ILE 1 12.963 1.638 -3.0451.00 0.00
ATOM 11 HG23 ILE 1 12.551 1.256 -4.7171.00 0.00
ATOM 12 CDl ILE 1 10.735 -2.319 -3.8951.00 0.00
ATOM 13 HD11 ILE 1 10.871 -3.209 -3.3001.00 0.00
ATOM 14 HD12 ILE 1 10.472 -2.596 -4.9051.00 0.00
ATOM 15 HD13 ILE 1 9.945 -1.718 -3.4691.00 0.00
ATOM l6 C ILE 1 10.762 -1.195 -1.2321.00 0.00
ATOM 17 0 ILE 1 10.856 -2.402 -1.3341.00 0.00
ATOM 18 N ILE 1 13.205 -0.849 -1.4751.00 0.00
ATOM 19 HT1 ILE 1 13.115 -1.875 -1.3351.00 0.00
ATOM 20 HT2 ILE 1 13.599 -0.414 -0.6151.00 0.00
ATOM 21 HT3 ILE 1 13.839 -0.665 -2.2781.00 0.00
ATOM 22 N LEU 2 9.722 -0.643 -0.6671.00 0.00
ATOM 23 HN LEU 2 9.666 0.337 -0.5911.00 0.00
ATOM 24 CA LEU 2 8.619 -1.500 -0.1361.00 0.00
ATOM 25 HA LEU 2 8.809 -2.544 -0.3491.00 0.00
ATOM 26 CB LEU 2 8.630 -1.265 1.375 1.00 0.00
ATOM 27 HB1 LEU 2 7.626 -1.058 1.714 1.00 0.00
ATOM 28 HB2 LEU 2 9.269 -0.424 1.604 1.00 0.00
ATOM 29 CG LEU 2 9.156 -2.514 2.083 1.00 0.00
ATOM 30 HG LEU 2 9.725 -3.111 1.384 1.00 0.00
ATOM 31 CD1 LEU 2 10.056 -2.100 3.249 1.00 0.00
ATOM 32 HD11 LEU 2 11.090 -2.148 2.940 1.00 0.00
ATOM 33 HD12 LEU 2 9.897 -2.770 4.081 1.00 0.00
ATOM 34 H1713LEU 2 9.816 -1.091 3.548 1.00 0.00
ATOM 35 CD2 LEU 2 7.977 -3.332 2.615 1.00 0.00
ATOM 36 HD21 LEU 2 7.730 -3.000 3.613 1.00 0.00
ATOM 37 HD22 LEU 2 8.246 -4.378 2.640 1.00 0.00
ATOM 38 HD23 LEU 2 7.123 -3.195 1.968 1.00 0.00
ATOM 39 C LEU 2 7.279 -1.067 -0.7361.00 0.00
ATOM 40 0 LEU 2 7.213 -0.161 -1.5441.00 0.00
ATOM 41 N SER 3 6.209 -1.707 -0.3491:00 0.00
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WO 2005/000192 PCT/IL2004/000570
66
ATOM 42 HN SER 3 6.283 -2.435 0.304 1.000.00
ATOM 43 CA SER 3 4.875 -1.331 -0.898 1.000.00
ATOM 44 HA SER 3 4.861 -0.288 -1.173 1.000.00
ATOM 45 CB SER 3 4.700 -2.201 -2.142 1.000.00
ATOM 46 HB1 SER 3 5.077 -1.671 -3.007 1.000.00
ATOM 47 HB2 SER 3 3.655 -2.421 -2.286 1.000.00
ATOM 48 OG SER 3 5.414 -3.418 -1.967 1.000.00
ATOM 49 HG SER 3 4.796 -4.082 -1.654 1.000.00
ATOM 50 C SER 3 3.777 -1.630 0.126 1.000.00
ATOM 51 O SER 3 3.504 -2.771 0.442 1.000.00
ATOM 52 N ARG 4 3.145 -0.613 0.646 1.000.00
ATOM 53 HN ARG 4 3.380 0.300 0.375 1.000.00
ATOM 54 CA ARG 4 2.063 -0.839 1.649 1.000.00
ATOM 55 HA ARG 4 1.545 -1.766 1.443 1.000.00
ATOM 56 CB ARG 4 2.784 -0.921 3.001 1.000.00
ATOM 57 HBl ARG 4 2.113 -0.610 3.789 1.000.00
ATOM 58 HB2 ARG 4 3.648 -0.270 2.990 1.000.00
ATOM 59 CG ARG 4 3.234 -2.362 3.257 1.000.00
ATOM 60 HG1 ARG 4 4.312 -2.395 3.326 1.000.00
ATOM 61 HG2 ARG 4 2.906 -2.991 2.444 1.000.00
ATOM 62 CD ARG 4 2.627 -2.865 4.571 1.000.00
ATOM 63 HD1 ARG 4 1.720 -2.327 4.798 1.000.00
ATOM 64 HD2 ARG 4 3.341 -2.763 5.378 1.000.00
ATOM 65 NE ARG 4 2.321 -4.302 4.326 1.000.00
ATOM 66 HE ARG 4 1.389 -4.605 4.292 1.000.00
ATOM 67 CZ ARG 4 3.292 -5.157 4.155 1.000.00
ATOM 68 NH1 ARG 4 4.516 -4.825 4.463 1.000.00
ATOM 69 HH11ARG 4 4.710 -3.915 4.830 1.000.00
ATOM 70 HH12ARG 4 5.259 -5.481 4.333 1.000.00
ATOM 71 NH2 ARG 4 3.038 -6.344 3.677 1.000.00
ATOM 72 HH21ARG 4 2.100 -6.599 3.441 1.000.00
ATOM 73 HH22ARG 4 3.782 -7.000 3.546 1.000.00
ATOM 74 C ARG 4 1.081 0.336 1.636 1.000.00
ATOM 75 0 ARG 4 1.468 1.482 1.752 1.000.00
ATOM 76 N ARG 5 -0.187 0.062 1.495 1.000.00
ATOM 77 HN ARG 5 -0.480 -0.873 1.402 1.000.00
ATOM 78 CA ARG 5 -1.192 1.168 1.475 1.000.00
ATOM 79 HA ARG 5 -0.705 2.127 1.595 1.000.00
ATOM 80 CB ARG 5 -1.844 1.083 0.094 1.000.00
ATOM 81 HBl ARG 5 -2.917 1.041 0.204 1.000.00
ATOM 82 HB2 ARG 5 -1.499 0.192 -0.412 1.000.00
ATOM 83 CG ARG 5 -1.465 2.316 -0.727 1.000.00
ATOM 84 HG1 ARG 5 -0.748 2.908 -0.179 1.000.00
ATOM 85 HG2 ARG 5 -2.350 2.907 -0.918 1.000.00
ATOM 86 CD ARG 5 -0.848 1.876 -2.057 1.000.00
ATOM 87 HD1 ARG 5 -0.300 0.955 -1.931 1.000.00
ATOM 88 HD2 ARG 5 -0.202 2.651 -2.445 1.000.00
ATOM 89 NE ARG 5 -2.008 1.659 -2.965 1.000.00
ATOM 90 HE ARG 5 -2.795 2.241 -2.903 1.000.00
ATOM 91 CZ ARG 5 -1.977 0.695 -3.845 1.000.00
ATOM 92 NH1 ARG 5 -0.857 0.392 -4.441 1.000.00
ATOM 93 HH11ARG 5 -0.022 0.898 -4.225 1.000.00
ATOM 94 HH12ARG 5 -0.834 -0.347 -5.115 1.000.00
ATOM 95 NH2 ARG 5 -3.067 0.035 -4.127 1.000.00
ATOM 96 HH21ARG 5 -3.925 0.267 -3.670 1.000.00
ATOM 97 HH22ARG 5 -3.043 -0.704 -4.801 1.000.00
ATOM 98 C ARG 5 -2.236 0.954 2.575 1.000.00
ATOM 99 0 ARG 5 -2.260 -0.078 3.214 1.000.00
ATOM.100 N PRO 6 -3.068 1.946 2.758 1.000.00
ATOM 101 CA PRO 6 -4.149 1.876 3.807 1.000.00
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WO 2005/000192 PCT/IL2004/000570
67
ATOM 102 HA PRO 6 -3.740 1.628 4.790 1.00 0.00
ATOM 103 CB PRO 6 -4.710 3.304 3.802 1.00 0.00
ATOM 104 HB1 PRO 6 -4.213 3.918 4.537 1.00 0.00
ATOM 105 HB2 PRO 6 -5.780 3.296 3.976 1.00 0.00
ATOM 106 CG PRO 6 -4.411 3.814 2.426 1.00 0.00
ATOM 107 HG1 PRO 6 -4.357 4.887 2.428 1.00 0.00
ATOM 108 HG2 PRO 6 -5.177 3.477 1.740 1.00 0.00
ATOM 109 CD PRO 6 -3.087 3.236 2.027 1.00 0.00
ATOM 110 HD2 PRO 6 -3.044 3.093 0.948 1.00 0.00
ATOM 111 HD1 PRO 6 -2.275 3.877 2.353 1.00 0.00
ATOM 112 C PRO 6 -5.282 0.893 3.432 1.00 0.00
ATOM 113 0 PRO 6 -6.393 1.035 3.902 1.00 0.00
ATOM 114 N SER 7 -5.030 -0.0932.607 1.00 0.00
ATOM 115 HN SER 7 -4.145 -0.2072.235 1.00 0.00
ATOM 116 CA SER 7 -6.110 -1.0512.233 1.00 0.00
ATOM 117 HA SER 7 -5.833 -1.6031.348 1.00 0.00
ATOM 118 CB SER 7 -6.238 -2.0013.415 1.00 O.OD
ATOM 119 HB1 SER 7 -6.552 -2.9723.057 1.00 0.00
ATOM 120 HB2 SER 7 -6.974 -1.6194.102 1.00 0.00
ATOM 121 OG SER 7 -4.984 -2.1044.077 1.00 0.00
ATOM 122 HG SER 7 -5.045 -2.8144.720 1.00 0.00
ATOM 123 C SER 7 -7.430 -0.3162.010 1.00 0.00
ATOM 124 0 SER 7 -8.251 -0.2112.899 1.00 0.00
ATOM 125 N TYR 8 -7.643 0.184 0.831 1.00 0.00
ATOM 126 HN TYR 8 -6.966 0.078 0.127 1.00 0.00
ATOM 127 CA TYR 8 -8.925 0.904 0.559 1.00 0.00
ATOM 128 HA TYR 8 -9.535 0.924 1.451 1.00 0.00
ATOM 129 CB TYR 8 -8.533 2.329 0.179 1.00 0.00
ATOM 130 HB1 TYR 8 -9.278 2.738 -0.498 1.00 0.00
ATOM 131 HB2 TYR 8 -7.570 2.317 -0.324 1.00 0.00
ATOM 132 CG TYR 8 -8.466 3.172 1.458 1.00 0.00
ATOM 133 CD1 TYR 8 -7.422 4.091 1.648 1.00 0.00
ATOM 134 HD1 TYR 8 -6.664 4.205 0.901 1.00 0.00
ATOM 135 CD2 TYR 8 -9.451 3.035 2.465 1.00 0.00
ATOM 136 HD2 TYR 8 -10.261 2.331 2.352 1.00 0.00
ATOM 137 CE1 TYR 8 -7.360 4.861 2.815 1.00 0.00
ATOM 138 HEl TYR 8 -6.553 5.566 2.952 1.00 0.00
ATOM 139 CE2 TYR 8 -9.379 3.809 3.629 1.00 0.00
ATOM 140 HE2 TYR 8 -10.134 3.702 4.394 1.00 0.00
ATOM 141 CZ TYR 8 -8.336 4.721 3.803 1.00 0.00
ATOM 142 OH TYR 8 -8.270 5.483 4.951 1.00 0.00
ATOM 143 HH TYR 8 -7.345 5.662 5.136 1.00 0.00
ATOM 144 C TYR 8 -9.680 0.230 -0.587 1.00 0.00
ATOM 145 0 TYR 8 -9.400 0.456 -1.747 1.00 0.00
ATOM 146 N ARG 9 -10.639 -0.592-0.266 1.00 0.00
ATOM 147 HN ARG 9 -10.848 -0.7510.681 1.00 0.00
ATOM 148 CA ARG 9 -11.423 -1.283-1.334 1.00 0.00
ATOM 149 HA ARG 9 -11.870 -0.561-1.999 1.00 0.00
ATOM 150 CB ARG 9 -10.408 -2.140-2.099 1.00 0.00
ATOM 151 HB1 ARG 9 -9.528 -1.554-2.314 1.00 0.00
ATOM 152 HB2 ARG 9 -10.849 -2.477-3.027 1.00 0.00
ATOM 153 CG ARG 9 -10.015 -3.354-1.253 1.00 0.00
ATOM 154 HG1 ARG 9 -10.141 -3.120-0.206 1.00 0.00
ATOM 155 HG2 ARG 9 -8.982 -3.606-1.444 1.00 0.00
ATOM 156 CD ARG 9 -10.907 -4.543-1.618 1.00 0.00
ATOM 157 HD1 ARG 9 -11.932 -4.342-1.351 1.00 0.00
ATOM 158 HD2 ARG 9 -10.556 -5.439-1.125 1.00 0.00
ATOM 159 NE ARG 9 -10.781 -4.677-3.096 1.00 0.00
ATOM 160 HE ARG 9 -11.439 -4.252-3.684 1.00 0.00
ATOM 161 CZ ARG 9 -9.794 -5.361-3.607 1.00 0.00
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68
ATOM 162 NH1 ARG 9 -8.770 -5.684 -2.865 1.000.00
ATOM 163 HH11ARG 9 -8.742 -5.408 -1.904 1.000.00
ATOM 164 HH12ARG 9 -8.014 -6.208 -3.256 1.000.00
ATOM 165 NH2 ARG 9 -9.831 -5.723 -4.860 1.000.00
ATOM 166 HH21ARG 9 -10.615 -5.476 -5.429 1.000.00
ATOM 167 HH22AR.G 9 -9.074 -6.247 -5.252 1.000.00
ATOM 168 C ARG 9 -12.504 -2.167 -0.705 1.000.00
ATOM 169 OT1 ARG 9 -13.492 -2.425 -1.372 1.000.00
ATOM 170 OT2 ARG 9 -12.324 -2.570 0.433 1.000.00
END
CA 02530111 2005-12-20
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69
Table 3
REMARK FILENAME="refine_1 20.pdb"
REMARK
REMARKoverall,bonds,angles,improper,vdw,noe,cdih
REMARKenergies: 104.733, 95, 767, 51384, .64866,
3.472 70.7 3. 6
20.3204,
$CDIH
REMARK
REMARKbonds,
angles,impropers,noe,cdih
REMARKrms-d: 652,0.496579,7.90724E-02,0
4.442149E-03,1.21
REMARK
REMARK noe,
cdih
REMARKviolations.: 0
0,
REMARK
REMARKDATE:03-Apr-00 08:41:00c reatedby user:orish
ATOM 1 CA ILE 1B -9.783-1.457-0.558 1.00 0.00
ATOM 2 HA ILE 1B -9.677-0.665-1.298 1.00 0.00
ATOM 3 CB ILE 1B -11.259-1.637-0.199 1.00 0.00
ATOM 4 HB TLE 1B -11.578-0.7960.417 1.00 0.00
ATOM 5 CG1 TLE 1B -11.441-2.9450.598 1.00 0.00
ATOM 6 HG11ILE 1B -12.251-2.8161.316 1.00 0.00
ATOM 7 HG12ILE 1B -10.519-3.1671.135 1.00 0.00
ATOM 8 CG2 ILE 1B -12.101-1.660-1.481 1.00 0.00
ATOM 9 HG21TLE 1B -12.492-0.662-1.677 1.00 0.00
ATOM 10 HG22ILE 1B -12.930-2.357-1.358 1.00 0.00
ATOM 11 HG23ILE 1B -11.480-1.978-2.318 1.00 0.00
ATOM 12 CD1 TLE 1B -11.776-4.119-0.334 1.00 0.00
ATOM 13 HD11ILE 1B -11.998-5.0040.263 1.00 0.00
ATOM 14 HI712ILE 1B -10.926-4.325-0.983 1.00 0.00
ATOM 15 HD13ILE 1B -12.644-3.866-0.941 1.00 0.00
ATOM 16 C ILE 1B -8.973-1.1370.677 1.00 0.00
ATOM 17 0 ILE 1B -9.510-0.7871.709 1.00 0.00
ATOM 18 N ILE 1B -9.351-2.764-1.130 1.00 0.00
ATOM 19 HTl ILE 1B -8.379-2.681-1.489 1.00 0.00
ATOM 20 HT2 ILE 1B -9.987-3.028-1.910 1.00 0.00
ATOM 21 HT3 ILE 1B -9.383-3.494-0.391 1.00 0.00
ATOM 22 N LEU 2 -7.676-1.2500.593 1.00 0.00
ATOM 23 HN LEU 2 -7.221-1.531-0.230 1.00 0.00
ATOM 24 CA LEU 2 -6.745-0.9701.725 1.00 0.00
ATOM 25 HA LEU 2 -7.286-0.6592.617 1.00 0.00
ATOM 26 CB LEU 2 -6.051-2.3051.992 1.00 0.00
ATOM 27 HB1 LEU 2 -5.606-2.6751.069 1.00 0.00
ATOM 28 HB2 LEU 2 -6.782-3.0272.359 1.00 0.00
ATOM 29 CG LEU 2 -4.955-2.1103.041 1.00 0.00
ATOM 30 HG LEU 2 -5.142-1.1903.595 1.00 0.00
ATOM 31 CDl LEU 2 -4.955-3.2964.007 1.00 0.00
ATOM 32 HD11LEU 2 -5.261-2.9584.997 1.00 0,00
ATOM 33 HD12LEU 2 -3.952-3.7204.062 1.00 0.00
ATOM 34 HD13LEU 2 -5.651-4.0553.651 1.00 0.00
ATOM 35 CD2 LEU 2 -3.595-2.0202.345 1.00 0.00
ATOM 36 HD21LEU 2 -3.732-1.6711.322 1.00 0.00
ATOM 37 HD22LEU 2 -3.127-3.0042.334 1.00 0.00
ATOM 38 HD23LEU 2 -2.956-1.3202.884 1.00 0.00
ATOM 39 C LEU 2 -5.7250.080 1.347 1.00 a.00
ATOM 40 0 LEU 2 -4.9150.491 2.155 1.00 0.00
ATOM 41 N SER 3 -5.7470.528 0.122 1.00 0.00
CA 02530111 2005-12-20
WO 2005/000192 PCT/IL2004/000570
ATOM 42 HN SER 3 -6.390 0.212 -0.547 1.00 0.00
ATOM 43 CA SER 3 -4.806 1.562 -0.398 1.00 0.00
ATOM 44 HA SER 3 -4.771 1.557 -1.486 1.00 0.00
ATOM 45 CB SER 3 -5.388 2.889 0.083 1.00 0.00
ATOM 46 HB1 SER 3 -6.272 3.133 -0.510 1.00 0.00
ATOM 47 HB2 SER 3 -4.648 3.677 -0.034 1.00 0.00
ATOM 48 OG SER 3 -5.738 2.778 1.457 1.00 0.00
ATOM 49 HG SER 3 -6.250 3.554 1.696 1.00 0.00
ATOM 50 C SER 3 -3.416 1.369 0.164 1.00 0.00
ATOM 51 0 SER 3 -3.131 1.747 1.283 1.00 0.00
ATOM 52 N ARG 4 -2.534 0.782 -0.597 1.00 0.00
ATOM 53 HN ARG 4 -2.747 0.466 -1.515 1.00 0.00
ATOM 54 CA ARG 4 -1.116 0.522 -0.175 1.00 0.00
ATOM 55 HA ARG 4 -0.840 1.104 0.716 1.00 0.00
ATOM 56 CB ARG 4 -1.095 -0.9750.176 1.00 0.00
ATOM 57 HB1 ARG 4 -1.739 -1.526-0.506 1.00 0.00
ATOM 58 HB2 ARG 4 -1.453 -1.1141.200 1.00 0.00
ATOM 59 CG ARG 4 0.323 -1.5210.077 1.00 0.00
ATOM 60 HG1 ARG 4 1.018 -0.714-0.142 1.00 0.00
ATOM 61 HG2 ARG 4 0.369 -2.273-0.711 1.00 0.00
ATOM 62 CD ARG 4 0.681 -2.1461.415 1.00 0.00
ATOM 63 HD1 ARG 4 -0.203 -2.6041.849 1.00 0.00
ATOM 64 HD2 ARG 4 1.067 -1.3732.079 1.00 0.00
ATOM 65 NE ARG 4 1.715 -3.1691.096 1.00 0.00
ATOM 66 HE ARG 4 2.519 -3.1001.652 1.00 0.00
ATOM 67 CZ ARG 4 1.576 -4.0750.168 1.00 0.00
ATOM 68 NH1 ARG 4 1.048 -5.2330.460 1.00 0.00
ATOM 69 HHTl ARG 4 0.750 -5.4241.395 1.00 0.00
ATOM 70 HH12 ARG 4 0.942 -5.927-0.252 1.00 0.00
ATOM 71 NH2 ARG 4 1.965 -3.825-1.052 1.00 0.00
ATOM 72 HH21 ARG 4 2.370 -2.938-1.276 1.00 0.00
ATOM 73 HH22 ARG 4 1.859 -4.520-1.764 1.00 0.00
ATOM 74 C ARG 4 -0.156 0.835 -1.306 1.00 0.00
ATOM 75 0 ARG 4 0.292 -0.044-2.015 1.00 0.00
ATOM 76 N ARG 5 0.150 2.088 -1.507 1.00 0.00
ATOM 77 HN ARG 5 -0.225 2.805 -0.970 1.00 0.00
ATOM 78 CA ARG 5 1.062 2.546 -2.605 1.00 0.00
.
ATOM 79 HA ARG 5 1.447 1.693 -3.157 1.00 0.00
ATOM 80 CB ARG 5 0.167 3.349 -3.540 1.00 0.00
ATOM 81 HB1 ARG 5 0.684 3.496 -4.489 1.00 0.00
ATOM 82 HB2 ARG 5 -0.044 4.319 -3.089 1.00 0.00
ATOM 83 CG ARG 5 -1.142 2.596 -3.784 1.00 0.00
ATOM 84 HGl ARG 5 -1.832 3.235 -4.334 1.00 0.00
ATOM 85 HG2 ARG 5 -1.587 2.319 -2.828 1.00 0.00
ATOM 86 CD ARG 5 -0.861 1.333 -4.602 1.00 0.00
ATOM 87 HD1 ARG 5 -1.790 0.801 -4.798 1.00 0.00
ATOM 88 HD2 ARG 5 -0.168 0.687.-4.058 1.00 0.00
ATOM 89 NE ARG 5 -0.259 1.834 -5.868 1.00 0.00
ATOM 90 HE ARG 5 0.592 1.404 -6.094 1.00 0.00
ATOM 91 CZ ARG 5 -0.804 2.756 -6.613 1.00 0.00
ATOM 92 NH1 ARG 5 -2.099 2.919 -6.607 1.00 0.00
ATOM 93 HH11 ARG 5 -2.673 2.338 -6.031 1.00 0.00
ATOM 94 HH12 ARG 5 -2.516 3.626 -7.178 1.00 0.00
ATOM 95 NH2 ARG 5 -0.054 3.515 -7.365 1.00 0.00
ATOM 96 HH21 ARG 5 0.938 3.389 -7.370 1.00 0.00
.
ATOM 97 HH22 ARG 5 -0.472 4.221 -7.936 1.00 0.00
ATOM 98 C ARG 5 2.235 3.432 -2.17 1.00 0.00
6
ATOM 99 0 ARG 5 3.149 3.598 -2.959 1.00 0.00
ATOM 100 N PRO 6 2.225 4.009 -0.990 1.00 0.00
ATOM 101 CA PRO & 3.362 4.885 -0.604 1.00 0.00
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ATOM 102 HA PRO 6 3.562 5.623 -1.380 1.00 0.00
ATOM 103 CB PRO 6 2.877 5.579 0.665 1.00 0.00
ATOM 104 HB1 6 2.405 6.534 0.423 1.00 0.00
PRO
ATOM 105 HB2 PRO 6 3.704 5.730 1.362 1.00 0.00
ATOM 106 CG PRO 6 1.867 4.642 1.236 1.00 0.00
ATOM 107 HG1 PRO 6 1.119 5.192 1.780 1.00 0.00
ATOM 108 HG2 PRO 6 2.357 3.932 1.887 1.00 0.00
ATOM 109 CD PRO 6 1.228 3.922 0.080 1.00 0.00
ATOM 110 HD2 PRO 6 1.038 2.890 0.343 1.00 0.00
ATOM 111 HDl PRO 6 0.316 4.415 -0.219 1.00 Ø00
ATOM 112 C PRO 6 4.587 4.040 -0.334 1.00 0.00
ATOM 113 0 PRO 6 5.195 4.120 0.714 1.00 0.00
ATOM 114 N SRP 7 4.973 3.235 -1.287 1.00 0.00
ATOM 115 HN SRP 7 4.501 3.180 -2.153 1.00 0.00
ATOM 116 CA SRP 7 6.178 2.345 -1.198 1.00 0.00
ATOM 117 C SRP 7 5.986 1.217 -0.187 1.00 0.00
ATOM 118 0 SRP 7 6.890 0.401 0.000 1.00 0.00
ATOM 119 CB SRP 7 7.409 3.196 -0.806 1.00 0.00
ATOM 120 OG1 SRP 7 7.614 4.247 -1.816 1.00 0.00
ATOM 121 PG2 SRP 7 9.111 4.826 -1.692 1.00 0.00
ATOM 122 OG3 SRP 7 9.841 4.741 -3.126 1.00 0.00
ATOM 123 OG2 SRP 7 9.883 4.016 -0.692 1.00 0.00
ATOM 124 OG4 SRP 7 9.056 6.362 -1.210 1.00 0.00
ATOM 125 HA SRP 7 6.354 1.890 -2.170 1.00 0.00
ATOM 126 HG3 SRP 7 10.770 4.312 -3.012 1.00 0.00
ATOM 127 HG4 SRP 7 8.124 6.751 -1.413 1.00 0.00
ATOM 128 HB1 SRP 7 8.290 2.553 -0.751 1.00 0.00
ATOM 129 HB2 SRP 7 7.240 3.659 0.163 1.00 0.00
ATOM 130 N TYR 8 4.845 1.147 0.452 1.00 0.00
ATOM 131 HN TYR 8 4.121 1.772 0.312 1.00 0.00
ATOM 132 CA TYR 8 4.521 0.098 1.463 1.00 0.00
ATOM 133 HA TYR 8 4.788 0.421 2.466 1.00 0.00
ATOM 134 CB TYR 8 3.001 -0.0611.371 1.00 0.00
ATOM 135 HB1 TYR 8 2.757 -1.1151.255 1.00 0.00
ATOM 136 HB2 TYR 8 2.631 0.495 0.510 1.00 0.00
ATOM 137 CG TYR 8 2.351 0.471 2.630 1.00 0.00
ATOM 138 CD1 TYR 8 2.895 0.164 3.884 1.00 0.00
ATOM 139 HD1 TYR 8 3.776 -0.4533.953 1.00 0.00
ATOM 140 CD2 TYR 8 1.202 1.269 2.544 1.00 0.00
ATOM 141 HD2 TYR 8 0.776 1.504 1.579 1.00 0.00
ATOM 142 CEl TYR 8 2.293 0.655 5.048 1.00 0.00
ATOM 143 HE1 TYR 8 2.713 0.417 6.014 1.00 0.00
ATOM 144 CE2 TYR 8 0.600 1.759 3.710 1.00 0.00
ATOM 145 HE2 TYR -0.284 2.374 3.643 1.00 0.00
8
ATOM 146 CZ TYR 8 1.146 1.452 4.961 1.00 0.00
ATOM 147 OH TYR 8 0.553 1.936 6.110 1.00 0.00
ATOM 148 HH TYR 8 1.222. 2.407 6.613 1.00 0.00
ATOM 149 C TYR 8 5.198 -1.2171.126 1.00 0.00
ATOM 150 0 TYR 8 5.057 -1.7280.033 1.00 0.00
ATOM 151 N ARG 9 5.936 -1.7882.046 1.00 0.00
ATOM 152 HN ARG 9 6.065 -1.3972.951 1.00 0.00
ATOM 153 CA ARG 9 6.655 -3.0951.832 1.00 0.00
ATOM 154 HA ARG 9 5.958 -3.9011.569 1.00 0.00
ATOM 155 CB ARG 9 7.597 -2.8470.639 1.00 0.00
ATOM 156 HB1 ARG 9 7.078 -2.256-0.115 1.00 0.00
ATOM 157 HB2 ARG 9 7.886 -3.8050.204 1.00Ø00
ATOM 158 CG ARG 9 8.858 -2.0971.089 1.00 0.00
ATOM 159 HGl ARG 9 8.772 -1.8252.139 1.00 0.00
ATOM 160 HG2 ARG 9 8.975 -1.1930.489 1.00 0.00
ATOM 161 CD ARG 9 10.085 -2.9960.895 1.00 0.00
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ATOM 162 HD1 ARG 9 10.070 -3.810 1.617 1.00 0.00
ATOM 163 HD2 ARG 9 10.998 -2.408 1.013 1.'000.00
ATOM 164 NE ARG 9 9.950 -3.518 -0.4931.00 0.00
ATOM 165 HE ARG 9 9.658 -4.453 -0.5341.00 0.00
ATOM 166 CZ ARG 9 10.193 -2.808 -1.5611.00 0.00
ATOM 167 NH1 ARG 9 11.414 -2.436 -1.8331.00 0.00
ATOM 168 HH11ARG 9 12.163 -2.695 -1.2231.00 0.00
ATOM 169 HH12ARG 9 11.600 -1.892 -2.6511.00 0.00
ATOM 170 NH2 ARG 9 9.215 -2.471 -2.3571.00 0.00
ATOM 171 HH21ARG 9 8.280 -2.756 -2.1491.00 0.00
ATOM 172 HH22ARG 9 9.402 -1.927 -3.1751.00 0.00
ATOM 173 C ARG 9 7.449 -3.492 3.057 1.00 0.00
ATOM 174 QTl ARG 9 7.344 -2.801 4.057 1.00 0.00
ATOM 175 OT2 A12G 9 8.155 -4.485 2.985 1.00 0.00
END
It is appreciated that certain features of the invention, which are, for
clarity,
described in the context of separate embodiments, may also be provided in
combination in a single embodiment. Conversely, various features of the
invention,
which are, for brevity, described in the context of a single embodiment, may
also be
provided separately or in any suitable subcombination.
Although the invention has been described in conjunction with specific
l0 embodiments thereof, it is evident that many alternatives, modifications
and variations
will be apparent to those skilled in the art. Accordingly, it is intended to
embrace all
such alternatives, modifications and variations that fall within the spirit
and broad
scope of the appended claims. A11 publications, patents and patent
applications
mentioned in this specification are herein incorporated in their entirety by
reference
into the specification, to the same extent as if each individual publication,
patent or
patent application was specifically and individually indicated to be
incorporated
herein by reference. In addition, citation or identification of any reference
in this
application shall not be construed as an admission that such reference is
available as
prior art to the present invention.
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73
REFERENCES
Aberle H, Bauer A, Stappert J, K.ispert A and Kemler R, "beta-catenin is a
target for the ubiquitin-proteasome pathway", EMBO J, 16:3797-802 (1997)
American Diabetes Association, "Standards of Medical Care for Patients With
Diabetes Mellitus", 21 Diabetes Care (1998).
Barber AJ, Nakamura M, Wolpert EB, et al Insulin Rescues Retinal Neurons
from Apoptosis by a Phosphatidylinositol 3-Kinase/Akt-mediated Mechanism That
Reduces the Activation of Caspase-3. JBiol Chem 276:32814-821 (2001).
Beasley C, Cotter D, Khan N, Pollard C, Sheppard P, Varndell I, Lovestone S,
Anderton B and Everall I, "Glycogen synthase kinase-3 beta immunoreactivity is
reduced in the prefrontal cortex in schizophrenia" Neut osci Lett 302:117-20
(2001)
Behrense J, Von Kries JP, Kuhl M, Bruhn L, Weldlich D, Grosschedl R and
Birchmeier W, "Functional interaction of beta-catenin with the transcription
factor
LEF-1" Nature, 382"638-42 (1996)
Berridge MJ, Downes CP and Hanley MR, "Neural and developmental actions
of lithium: a unifying hypothesis", Cell 59:411-419 (1989)
Bhat RV and Budd SL "GSK-3 beta signaling" casting a wide net in
Alzheimer's disease" Neurosig~tals 11:251-61 (2002)
Bijur GN, De Sarno P, RS. J Glycogen synthase kinase-3 beta facilitates
staurosporine- and heat shock-induced apoptosis. Protection by lithium J Biol
Claem
275:7583-90 {2000)
Bradford MM, Anal Biochem 72:248-254 (1976)
Burke et al, "4'-O-[2-(2-fluoromalonyl))-L-tyrosine: a phosphotyrosyl mimic
for the preparation of signal transduction inhibitory peptides", J Med Chem
39(5):1021-1027 (1996a)
Burke et al, "Nonhydrolyzable phosphotyrosyl mimetics for the preparation of
phosphatase-resistant SH2 domain inhibitors", Biochemistry 33(21):6490-6494
( 1994a)
Burke et al, "Potent inhibition of insulin receptor dephosphorylation by a
hexamer peptide containing the phosphotyrosyl mimetic F2Pmp", Biochem Biophys
Res Commuu 204(1):129-133 (1994b)
Burke et al, "Small molecule interactions with protein-tyrosine phosphatase
PTP1B and their use in inhibitor design", Bioclaemistry 35(50):15989-15996
(1996b)
CA 02530111 2005-12-20
WO 2005/000192 PCT/IL2004/000570
74
Chen et al, "Why is phosphonodifluoromethyl phenylalanme a more potent
inhibitory moiety than phosphonomethyl phenylalanine toward protein-tyrosine
phosphatases?", Biochem Biophys Res Commu~c 216(3):976-984 (1995)
Chen G, Huang LD, Jiang YM and Manji HK "The mood-stabilizeing agent
valproate inhibits the activity of glycogen synthase kinase-1" J Neurochem
72:1327
30 (1999)
Cheng K, Creacy S, Lamer J Insulin-like effect of lithium ion on isolated rat
adipocytes stimulation of glycogenesis beyond glucose transport. Mol. Cell.
Biochem.
56:177-182 (1983)
Cheng, K., Creacy, S. & Lamer, J. Molecular & Cellular Biochemistry 56,
183-9 (1983)
Cheng, K., Creacy, S. & Lamer, J. Molecular ~ Cellular Biocheynistry 56,
177-82 (1983)
Chu et al, "Sequential phosphorylation by mitogen-activated protein kinase
and glycogen synthase kinase 3 represses transcriptional activation by heat
shock
factor-1",JBiolChem 271(48):30847-30857 (1996)
Coghlan, M. P., Culbert, A. A., Cross, D. A., Corcoran, S. L., Yates, J. W.,
Pearce, N. J., Rausch, O. L., Murphy, G. J., Carter, P. S., Roxbee Cox, L.,
Mills, D.,
Brown, M. J., Haigh, D., Ward, R. W., Smith, D. G., Murray, K. J., Reith, A.
D. &
Holder, J. C. Chemistry & Biology 7, 793-803 (2000)
Cohere, P. Muscle glycogen synthase, The enzymes, edited by Boyer. P, and
Krebs, E. G. (Academic Press, Orlando, FL) (1986)
Coyle-Rink L, Del Valle L, Sweet T, Khalili K and Amini S, "development
expression of Wnt signaling factors in mouse brain" Cancer Biol Ther 1:640-S
(2002)
Cross D.A., Culbert A.A., Chalmers K.A., Facci L., Skaper S.D., Reith, A.D. J
Neurochem 77:94-102 (2001).
Cross et al, "Inhibition of glycogen synthase kinase-3 by insulin mediated by
protein kinase B", Nature 378(6559):785-78 (1995)
Cross, D. A., Alessi, D. R., Vandenheede, J. R., McDowell, H. E., Hundal, H.
S. & Cohere, P. Biochem. J. 303, 21-26 (1994)
Crowder RJ, RS. F Glycogen synthase kinase-3 beta activity is critical for
neuronal death caused by inhibiting phosphatidylinositol 3-kinase or Akt but
not for
death caused by nerve growth factor withdrawal. JBiol Chem 275:34266-71 (2000)
CA 02530111 2005-12-20
WO 2005/000192 PCT/IL2004/000570
Dajani et al., "Crystal structure of glycogen synthase kinase 3~3: structural
basis for phosphate-primed substrate specificity and auto inhibition", Cell
105:721-
732 (2001)
Damiens, E., Baratte, B., Marie, D., Eisenbrand, G. & Meijer, L. Ohcogene
5 20, 3786-97 (2001)
Damsbo, P., Vaa.g, A., Hother-Nielsen, O. & Beck-Nielsen, H. Diabetologia
34, 239-45 {1991)
Davies, S. P., Reddy, H., Caivano, M. & Cohen, P. Biochemical Journal 351,
95-105 (2000)
10 Devlin, Textbook of'Biochemistry with Clinical Correlatio~ts, 4th Ed.
(Wiley-
Liss, Inc., 1997)
Degas et al, Bioorgahic Chemistry (Springer-Verlag, New York, 1981), pp 54-
92
Eldar-Finkelman et al, "Expression and characterization of glycogen synthase
15 kinase-3 mutants and their effect on glycogen synthase activity in intact
cells", Proc
Natl Acad Sci USA 93(19):10228-10233 (1996)
Eldar-Finkelman et al, "Increased glycogen synthase kinase-3 activity in
diabetes- and obesity-prone C57BL/6J mice", Diabetes 48(8):1662-1666 (1999)
Eldar-Finkelman et al, "Phosphorylation of insulin receptor substrate 1 by
20 glycogen synthase kinase 3 impairs insulin action", Proc Natl Acad Sci USA
94(18):9660-9664 (1997)
Eldar-Finkehnan, H. & Krebs, E. G. Proc .Natl. Acad. Sci. 94, 9660-9664
(1997)
Eldar-Finkelman, H. Tread. Mol. Med. 8, 126-132 (2002)
25 Eldar-Finkelman, H., Agrast, G. M., Foord, O., Fischer, E. H. & Krebs, E.
G.
Proc. Natl. Acad. Sci. USA 93, 10228-10233 {1996)
Eldar-Finkelman, H., Schreyer, S. A., Shinohara, M. M., LeBoeuf, R. C. &
Krebs, E. G. Diabetes 48, 1662-1666 {1999)
Emoto, M., Langille, S. E. & Czech, M. P. J. Biol. Chem. 276, 10677-82
30 (2001)
Fiol. et al, "A secondary phosphorylation of CREB341 at Serl29 is required
for the cAMP-mediated control of gene expression. A role for glycogen synthase
kinase-3 in the control of gene expression", JBiol Chem 269(51):32187-32193
(1994)
CA 02530111 2005-12-20
WO 2005/000192 PCT/IL2004/000570
76
Fiol et al, "Formation of protein kinase recognition sites by covalent
modification of the substrate. Molecular mechanism for the synergistic action
of
casein kinase II and glycogen synthase kinase 3", J Biol Chem 262(29):14042-
14048
(1987)
Fiol et al, "Ordered multisite protein phosphorylation. Analysis of glycogen
synthase kinase 3 action using model peptide substrates", JBiol Chem
265(11):6061-
6065 (1990)
Fiol et al, "Phosphoserine as a recognition determinant for glycogen synthase
kinase-3: phosphorylation of a synthetic peptide based on the G-component of
protein
1o phosphatase-1 Arch Biochem Biophys 267(2):797-802 (1988)
Fiol, C. J., Mahrenholz, A. M., Wang, Y., Roeske, R. W. & Roach, P. J.
(1987) J. Biol. Chem. 262, 14042-8.
Fu et al, Design and synthesis of a pyridone-based phosphotyrosine mimetic",
BioorgMed ChemLett 8(19):2813-2816 (1998)
Gao et al, "Inhibition of Grb2 SH2 domain binding by non-phosphate-
containing ligands. 2. 4-(2-Malonyl)phenylalanine as a potent phosphotyrosyl
mimetic", JMed Chem 43(5):911-920 (2000)
Gething et al, "Cell-surface expression of influenza haemagglutinin from a
cloned DNA copy of the RNA gene Nature 293(5834):620-625 (1981)
Grimes CA and Jope RS "The multifunctional roles of glycogen synthase
kinase 3 beta in cellular signaling" Prog Neurobiolo 65:391-426 (2001)
Groves et al, "Structural basis for inhibition of the protein tyrosine
phosphatase 1B by phosphotyrosine peptide mimetics", Biochemistry 37(51):17773-
17783 (1998)
Hallstrom et al, "Regulation of transcription factor Pdrlp function by an
Hsp70 protein in Saccharomyces cerevisiae", Mol Cell Biol 18(3):1147-1155
(1998)
Hanger DP, Hughes K, Woodgett JR, Brion JP, Anderton BH Glycogen
synthase kinase-3 induces Alzheimer's disease-like phosphorylation of
tau:.generation
of paired helical filament epitopes and neuronal localisation of the kinase.
Neurosci.
Lett. 147:58-62 (1992).
Hawiger J, "Cellular import of functional peptides to block intracellular
signaling Curr Opi~c Immunol 9(2):189-194 (1997)
Hawiger, J. Curs: Opin. Immun. 9, 189-194 (1997)
CA 02530111 2005-12-20
WO 2005/000192 PCT/IL2004/000570
77
Hawiger, J. (Cur Opi~c. Chem. Biol. 3, 89-94 1999}
He et al, Glycogen synthase kinase-3 and dorsoventral patterning in Xenopus
embryos", Nature 374(6523}:617-622 (1995)
Heinemann, L., Pfutzner, A. & Heise, T. Curr Pharm. Des. 14, 1327-1351
{2001 )
Herbst, J. J., Andrews, G. C., Contillo, L. G., Singleton, D. H., Genereux, P.
E., Gibbs, E. M. & Lienhard, G. E. J. Biolo.Chem. 270, 26000-5 (1995)
Hernandez F, Borrell J, Guaza C, Avila J and Lucas JJ "Spatila learning
deficit in transgenic mice that conditionally over-express GSK-3 beta in the
brain but
do not form tau filaments" JNeu~ohem 83:1529-33 (2002)
Higashimoto et al, "Human p53 is phosphorylated on serines 6 and 9 in
response to DNA damage-inducing agents", J Biol Chem 275(30):23199-23203
(2000)
Ikeda S, Kishida S, Yamamoto H. Murai H, Koyama S and Kikuchi A "Axin,
a negative regulator of the Wnt signaling pathway, forms a complex with GSK-3
beta
and beta-catenin and promotes GSK-3 beta-dependent phosphorylation of beta-
catenin" EMBO J 17:1371-84 {1998)
Jope RS and Bijur GN "Mood stabilizers, glycogen synthase kinase-3 beta
and cell survival" Molecular Psychiatry 7(1): 535-45 (2002)
Jung, T., Kamm, W., Breitenbach, A., Kaiserling, E., Xiao, J. X. & Kissel, T.
Euro. .I. Pharma. Biopharma. 50, 147-60 (2000)
Katagiri, H., Asano, T., Ishihara, H., Inukai, K., Shibasaki, Y., Kikuchi, M.,
Yazaki, Y. & Oka, Y. .I Biol Chem 271, 16987-90 (1996)
Klein PS, Melton DA "A Molecular Mechanism for the Effect of Lithium on
Development". Proc. Natl. Acad. Sci. USA 93:8455-8459 (1996).
Kole et al, "Protein-tyrosine phosphatase inhibition by a peptide containing
the
phosphotyrosyl mimetic, L-O-malonyltyrosine", Bioehem Biophys Res Commun
209(3):817-822 (1995)2
Kole et al, "Specific inhibition of insulin receptor dephosphorylation by a
synthetic dodecapeptide containing sulfotyrosyl residues as phosphotyrosyl
mimetic",
Iudiau .l Biochem Biophys 34(1-2):50-55 (1997)
CA 02530111 2005-12-20
WO 2005/000192 PCT/IL2004/000570
78
Latimer et al, "Stimulation of MAP kinase by v-raf transformation of
fibroblasts fails to induce hyperphosphorylation of transfected tau", FEBS
Lett
365:42-46 (1995)
Lawrence, J. C., Guinovart, J. J. & Lamer, J. J. Biol. Chem. 252, 444-450
s (1977)
Lovestone et al, CurrBiol4:1077-1086 (1995)
Lucas JJ, Hemandez F, Gomez-Ramos P, Moran MA, Hen R, J. A "Decreased
nuclear beta-catenin, tahyperphosphorylation and neurodegeneration in GSK-
3beta
conditional transgenic mice". EMBO J 20:27-39 (2001 )
Mandelkow EM, Drewes G, Biernat J, et al "Glycogen synthase kinase-3 and
the Alzheimer-like state of microtubule-associated protein tau". Febs Lett.
314:315-21
(1992)
Mandelkow et al, "Tau as a marker for Alzheimer's disease", Trends Biochem
Sci. 18(12):480-483 (1983)
i5 Manji et al, "Lithium at 50: have the neuroprotective effects of this
unique
cation been overlooked?", Biol Psyclzic~try 46(7):92,9-940 (1999)
Manji HK and Lenox RH "Signaling: cellular insights into the
pathophysiology of bipolar disorder" Biol. Psych. 48:518-30 (2001 )
Mauvais-Jarvis, F., Ueki, K., Fruman, D. A., Hirshman, M. F., Sakamoto, K.,
Goodyear, L. J., Iannacone, M., Accili, D., Cantley, L. C. & Kahn, C. R. J. .
Cliu.
Invest. 109, 141-9 (2002)
McKinsey et al, "Phosphorylation of the PEST domain of IkappaBbeta
regulates the function of NF-kappaB/lkappaBbeta complexes", J Biol Chem
272(36):22377-22380 (1997)
Merrifield et al, .IAm Chem Soc 85:2149 (1964)
Mikol et al, "The crystal structures of the SH2 domain of p561ck complexed
with two phosphonopeptides suggest a gated peptide binding site", J Mol Biol
246(2):344-355 (1995)
Miller JR and Moon RT "Signal transduction through beta-catenin and
specofocation of cell fate during embryogenesis" Ge~tes ~ development 10:2527-
39
(1996)
Morfini, G., Szebenyi, G., Elluru, R., Rather, N. & Brady, S. T. EMBD .I 21,
281-93 (2002)
CA 02530111 2005-12-20
WO 2005/000192 PCT/IL2004/000570
79
Mornson et al, Organic Chemistry, 6th Ed. (Prentice Hall, 1992)
Mulot et al, "Phosphorylation of tau by glycogen synthase kinase-3 beta in
vitro produces species with similar electrophoretic and immunogenic properties
to
PHF-tau from Alzheimer's disease brain", Biochem Soc Trans 23(1):455 (1995)
Mulot et al, "PHF-tau from Alzheimer's brain comprises four species on SDS-
PAGE which can be mimicked by in vitro phosphorylation of human brain tau by
glycogen synthase kinase-3 beta", FEBSLett 349(3):359-364 (1994)
Myers et al, "RS-1 activates phosphatidylinositol 3'-kinase by associating
with
src homology 2 domains of p85d", Proc Natl Acad Sci USA 89(21):10350-10354
(1992)
Nicolaou et al, "Design and synthesis of a peptidomimeticemploying (3-D-
glucose for scaffolding" in Peptides, Rivier and Marshall (eds) ESCOM (1990)
Nikoulina et al, "Potential role of glycogen synthase kinase-3 in skeletal
muscle insulin resistance of type 2 diabetes", Diabetes 49(2):263-271 (2000)
Nikoulina et al, "Regulation of glycogen synthase activity in cultured
skeletal
muscle cells from subjects with type II diabetes: role of chronic
hyperinsulinemia and
hyperglycemia", Diabetes 46(6):1017-1024 (1997)
Nonaka et al., Proc. Natl. Acad. Sci. USA, 95:2642-2647 (1998)
Otaka et al, Chem Commun (12):1081-1082 (2000)
Otaka et al, Tetrahedron Lett 36(6):927-30 (1995)
Pap M, Cooper G "Role of glycogen synthase kinase-3 in the
phosphatidylinositol 3-Kinase/Akt cell survival pathway". J. Biol.Chem.
273:19929-
32 (1998)
Peifer M and Polakis P ".Wnt signaling in oncogensis and embryogenesis - a
look outside the nucleus" Science 287:1606-9 (2000)
Phiel CJ, Klein PS "Molecular targets of lithium action". Annu Rev Pharmacol
Toxicol41:789-813 (2001)
Porsolt RD, Le Pichon M and Jalfre M "Depression: a new animal model
sensitive to antidepressant treatments" Nature 266:730-2 (1977)
Rich DH, in Protease Inhibitors, Barrett and Selveson (eds) Elsevier (1986)
Ricort, J. M., Tanti, J. F., Van Obberghen, E. & Le Marchand-Brustel, Y. Eur.
J. Biochem. 239, 17-22 (1996)
Rojas, M., Yao, S. & Lin, Y. Z. J. Biol.Chem. 271, 27456-61 {1996)
CA 02530111 2005-12-20
WO 2005/000192 PCT/IL2004/000570
~0
Roller et al, "Potent inhibition of protein-tyrosine phosphatase-1B using the
phosphotyrosyl mimetic fluoro-O-malonyl tyrosine (FOMT)", Bioorg Med Chem Lett
8(16):2149-2150 (1998)
Rubinfeld et al, "Binding of GSK3beta to the APC-beta-catenin complex and
regulation of compleR assembly", Science 272(5264):1023-1026 (1996)
Sakanaka C, Weiss JB and Williams LT "Bridging of beta-catenin and
glycogen synthase kinase-3 beta by axin and inhibition of beta-catenin
mediated
transcription" Proc Natl Acad Sci USA 95:3020-3 (1998)
Sakaue, H., Ogawa, W., Takata M, Kuroda S, Kotani K, Matsumoto M,
Sakaue M, Nishio S, Ueno, H. & Kasuga, M. K. Mol. Endocnin. 10, 1552-62 (1997)
Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Ed. (Cold
Spring Harbor Press, 1989)
Schiller et al, Int JPent Pot Rep 25:171 (1985)
Senel, S., Kremer, M., Nagy, K. & Squier, C. Cup. Pharm. Biotechnol. 2,
175-186(2001)
Shapiro et al, "Combined Fmoc-Alloc strategy for a general SPPS of
phosphoserine peptides; preparation of phosphorylation-dependent tau
antisera",
Bioo~gMed Chew 5(1):147-56 (1997)
Sherman et al, JAm Chem Soc 112:433 (i990)
Shulman et al, "Quantitation of muscle glycogen synthesis in normal subjects
and subjects with non-insulin-dependent diabetes by 13C nuclear magnetic
resonance
spectroscopy", NEngl JMed 322(4):223-228 (1990)
Stambolic V, Ruel L, Woodgett JR "Lithium inhibits glycogen synthase
kinase-3 activity and mimics wingless signalling in intact cells". Curr Biol.
6:1664-
1668 (1996).
Surwit, R. S., Kuhn, C. M., Cochrane, C., McCubbin, J. A. & Feinglos, M. N.
Diabetes 37, 1163-67 (1988)
Ter Haar et al., "Structure of GSK-3 beta reveals a primed phosphorylation
mechanism", Nat. Struct. Biol. 8(7):593-6 (2001)
Thomas, J. A., Schlender, K. K. & Lamer, J. Anal. Biochem. 25, 486-499
(1968)
Thomas, J: Am. Geniatr. Soc., 43:1279-89 (1995)
CA 02530111 2005-12-20
WO 2005/000192 PCT/IL2004/000570
81
Thorsett et al, "Dipeptide mimics. Conformationally restricted inhibitors of
angiotensin-converting enzyme", Bioclaem Biophys Res Commun 111(1):166-171
(1983)
Tong N, Sanchez JF, Maggirwar SB, et al "Activation of glycogen synthase
kinase 3 beta (GSK-3beta) by platelet activating factor mediates migration and
cell
death in cerebellar granule neurons". Eur.I Neurosci 13:1913-22 (2001)
Ueki, K., Yballe, C. M., Brachmann, S. M., Vicent, D., Watt, J. M., Kahn, C.
R. & Cantley, L. C. Proc. Natl. Acad. Sci. USA 99, 419-24 (2002)
Veber et al, "Conformationally restricted bicyclic analogs of somatostatin",
'roc Natl Acad Sci USA 75(6):2636-2640 (1978)
Wang, Y. & Roach, P. J. .I. Biol. Chem. 268, 23876-23880 (1993)
Welsh et al, "Glycogen synthase kinase-3 is rapidly inactivated in response to
insulin and phosphorylates eukaryotic initiation factor eIF-2B", Biochem .I
294(Pt
3):625-629 (1993)
Wiemann et al, Tetrahedron 56:1331-1337 (2000)
Woodgett, J. R. & Cohere, P. Biochim. Biophys. Acta. 788, 339-47 (1984)
Woodgett, J. R. Sci. STKE 100, RE12 (2001)
Ye et al, "L-O-(2-malonyl)tyrosine: a new phosphotyrosyl mimetic for the
preparation of Src homology 2 domain inhibitory peptides", .I Med Chem
38(21j:4270-4275 (1995)
Yost C, Torres M, Miller J, Huang E, IKimelman D and Moon R "The axis-
inducing activity, stability and subcellular disribution of beta-catenin is
regulated in
Xenopus embryos by glycogen synthase kinase 3" Genes 10:1443-1454 (1996)
Zasloff, M. Nature 415, 389-95 (2002)
Zhang, W., Depaoli-Roach, A. A. & Roach, P. J. Arch. Biochem. Biophys.
304, 219-25 (1993)
Zhang, Z. H., Johnson, J. A., Chen, L., El-Sherif, N., Mochly-Rosen, D. &
Boutjdir, M. Cifc. Res. 80, 720-9 (1997)
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1
SEQUENCE LISTING
<110> Eldar-Finkelman, Hagit
<120> GLYCOGEN SYNTHASE KINi~SE-3 INHIBITORS
<130> 26378
<160> 4
<170> PatentIn version 3.2
<210> 1
<211> 5
<212> PRT
<213> Artificial sequence
<220>
<223> GSK-3 recogi~.ition motif consensus
<220>
<221> misc feature
<222> (1) . . (1)
<223> Ser or Thr
<220>
<221> misc feature
<222> (2)..(4)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc feature
<222> (5)..(5)
<223> Phosphorylated Ser or Thr
<400> 1
Xaa Xaa Xaa Xaa Xaa
1 5
CA 02530111 2005-12-20
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2
<210> 2
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> Synthetic peptide
<220>
<221> MOD RES
<222> (7)..(7)
<223> PI30SPFi0RYLATION
<400> 2
Ile Leu Ser Arg Arg Pro Ser Tyr Arg
1 5
<210> 3
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> Synthetic peptide
<400> 3
Ile Leu Ser Arg Arg Pro Ser Tyr Arg
1 5
<210> 4
<211> 9
<212> PRT
<213> Artificial sequence
CA 02530111 2005-12-20
WO 2005/000192 PCT/IL2004/000570
<220>
<223> Synthetic peptide
<400> 4
Ile I~eu Ser Arg Arg Pro Glu Tyr Arg
