CA2530111A1 - Glycogen synthase kinase-3 inhibitors - Google Patents

Abstract

Compounds capable of inhibiting GSK-3 activity, pharmaceutical compositions including same and methods of using same in the treatment of GSK-3 mediated conditions are disclosed.

Description

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 itself, which phosphorylates insulin-receptor substrate-1 on serine residues (Eldar-Finkelman et al., 1997).

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;

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 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.

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.

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 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.

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.

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.

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 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;

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 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 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 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;

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 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 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, 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.

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" .

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 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 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.

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).

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.

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 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, 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).

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.

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 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.

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 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.

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 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 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 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 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;

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 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.

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 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 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.

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.

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.

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 and 9ECREB completely failed to be phosphorylated by GSK-3 (data not shown), 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 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 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 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, 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.

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 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, 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), 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 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 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), 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

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), 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).

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 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

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

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), 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 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 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 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 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 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.

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).

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 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

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 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'-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 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 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.

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 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 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 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

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 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 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 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 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 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.

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) 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) 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-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) 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-(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) 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) 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-(1996) Morfini, G., Szebenyi, G., Elluru, R., Rather, N. & Brady, S. T. EMBD .I 21, 281-93 (2002) 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) ~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) 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) 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 <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 <210> 3 <211> 9 <212> PRT
<213> Artificial sequence <220>
<223> Synthetic peptide <400> 3 Ile Leu Ser Arg Arg Pro Ser Tyr Arg <210> 4 <211> 9 <212> PRT
<213> Artificial sequence <220>
<223> Synthetic peptide <400> 4 Ile I~eu Ser Arg Arg Pro Glu Tyr Arg

Claims (185)

1. A compound having a general formula:

wherein:
X, Y, Z and W are each independently a carbon atom or a nitrogen atom;
A is alkyl or absent;

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, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, cyano, nitro, 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, hydrazine, aminoalkyl and a hydrophobic moiety; and R1, 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, nitro, 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, provided that at least one of X, Y, Z and W is a nitrogen atom and/or at least one of R1, R2, R3 and R4 is a group containing at least one amino moiety, and with the proviso that the compound is not pyridoxal phosphate.

2. The compound of claim 1, being capable of inhibiting an activity of GSK-3.

3. The compound of claim 1, wherein A is alkyl.

4. The compound of claim 1, wherein L is a phosphor atom.

5. The compound of claim 4, wherein each of Q, G and D is oxygen.

6. The compound of claim 4, wherein E is hydroxy.

7. The compound of claim 1, wherein at least one of X, Y, 2 and W is a nitrogen atom.

8. The compound of claim 7, wherein at least two of X, Y, Z and W are nitrogen atoms.

9. The compound of claim 8, wherein X and Y are each a nitrogen atom.

10. The compound of claim 8, wherein Z and W are each a nitrogen atom.

11. The compound of claim 1, wherein at least two of R1, R2, R3 and R4 are said groups containing at least one amino moiety.

12. The compound of claim 11, wherein each of R1 and R2 is said group containing at least one amino moiety.

13. The compound of claim 11, wherein each of R3 and R4 is said group containing at least one amino moiety.

14. The compound of claim 1, wherein D is a hydrophobic moiety.

15. The compound of claim 14, wherein said hydrophobic moiety is 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.

16. The compound of claim 15, wherein said fatty acid is selected from the group consisting of myristic acid, lauric acid, palmitic acid, stearic acid, oleic acid, arachidonic acid, linoleic acid and linolenic acid.

17. The compound of claim 5, wherein A is alkyl, each of X, Y, Z and W
is a carbon atom, and at least one of R3 and R4 is said group containing at least one amino moiety.

18. The compound of claims 1-17, wherein said at least one amino moiety is selected from the group consisting of guanidino, guanidinoalkyl, amino, aminoalkyl, hydrazine, guanyl, guanylinoalkyl, and any combination thereof.

19. The compound of claims 1-17, wherein said group containing at least one amino moiety comprises at least one positively charged group.

20. The compound of claim 19, wherein said at least one positively charged group comprises an ammonium ion.

21. The compound of claim 19, wherein said at least one positively charged group has a chemical structure derived from a side chain of a positively charged amino acid.

22. The compound of claim 21, wherein said positively charged amino acid is selected from the group consisting of arginine, lysine, histidine, proline and any derivative thereof.

23. The compound of claim 1, wherein each of X, Y and Z is a carbon atom and W is a nitrogen atom.

24. A compound having a general formula:
wherein:
X, Y, Z and W are each independently a carbon atom or a nitrogen atom;
A is alkyl or absent;
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 a hydrophobic moiety; and R1, 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, nitro, 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, hydrazine, aminoalkyl and an ammonium ion, or a pharmaceutically acceptable salt thereof.

25. The compound of claim 24, being capable of inhibiting an activity of GSK-3.

26. The compound of claim 24, wherein said hydrophobic moiety is 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.

27. The compound of claim 26, wherein said fatty acid is selected from the group consisting of myristic acid, lauric acid, palmitic acid, stearic acid, oleic acid, arachidonic acid, linoleic acid and linolenic acid.

28. The compound of claim 24, wherein A is alkyl.

29. The compound of claim 24, wherein L is a phosphor atom.

30. The compound of claim 29, wherein each of Q, G and D is oxygen.

31. The compound of claim 29, wherein E is hydroxy.

32. The compound of claim 24, wherein at least one of X, Y, Z and W is a nitrogen atom.

33. The compound of claim 32, wherein at least two of X, Y, Z and W are nitrogen atoms.

34. The compound of claim 33, wherein X and Y are each a nitrogen atom.

35. The compound of claim 33, wherein Z and W are each a nitrogen atom.

36. The compound of claim 32, wherein W is a nitrogen atom.

37. The compound of claim 24, wherein at least one of R1, R2, R3 and R4 is a group containing at least one amino moiety.

38. The compound of claim 37, wherein at least two of R1, R2, R3 and R4 are said groups containing at least one amino moiety.

39. The compound of claim 38, wherein each of R1 and R2 is said group containing at least one amino moiety.

40. The compound of claim 38, wherein each of R3 and R4 is said group containing at least one amino moiety.

41. The compound of claim 30, wherein A is alkyl.

42. The compound of claim 41, wherein each of X, Y, Z and W is a carbon atom.

43. The compound of claim 42, wherein each of R1, R2, R3 and R4 is hydrogen.

44. The compound of claim 41, wherein each of X, Y and Z is a carbon atom and W is a nitrogen atom.

45. The compound of claim 42, wherein at least one of R3 and R4 is a group containing at least one amino moiety.

46. The compound of claims 37-40 and 45, wherein said at least one amino moiety is selected from the group consisting of guanidino, guanidinoalkyl, amino, aminoalkyl, hydrazine, guanyl, guanylinoalkyl, and any combination thereof.

47. The compound of claims 37-40 and 45, wherein said.group containing at least one amino moiety comprises at least one positively charged group.

48. The compound of claim 47, wherein said at least one positively charged group comprises an ammonium ion.

49. The compound of claim 47, wherein said at least one positively charged group has a chemical structure derived from a side chain of a positively charged amino acid.

50. The compound of claim 49, wherein said positively charged amino acid is selected from the group consisting of arginine, lysine, histidine, proline and any derivative thereof.

51. A pharmaceutical composition comprising, as an active ingredient, a compound having the general formula:

wherein:
X, Y, Z and W are each independently a carbon atom or a nitrogen atom;
A is alkyl or absent;

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, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, cyano, nitro, 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 a hydrophobic moiety; and R1, 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, nitro, 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, said compound being capable of inhibiting an activity of GSK-3, and a pharmaceutically acceptable carrier.

52. The pharmaceutical composition of claim 51, wherein A is alkyl.

53. The pharmaceutical composition of claim 51, wherein L is a phosphor atom.

54. The pharmaceutical composition of claim 53, wherein each of Q, G
and D is oxygen.

55. The pharmaceutical composition of claim 54, wherein E is hydroxy.

56. The pharmaceutical composition of claim 51, wherein at least one of X, Y, Z and W is a nitrogen atom.

57. The pharmaceutical composition of claim 56, wherein at least two of X, Y, Z and W are nitrogen atoms.

58. The pharmaceutical composition of claim 57, wherein X and Y are each a nitrogen atom.

59. The pharmaceutical composition of claim 57, wherein Z and W are each a nitrogen atom.

60. The pharmaceutical composition of claim 51, wherein D is a hydrophobic moiety.

61. The pharmaceutical composition of claim 60, wherein said hydrophobic moiety is 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.

62. The pharmaceutical composition of claim 61, wherein said fatty acid is selected from the group consisting of myristic acid, lauric acid, palmitic acid, stearic acid, oleic acid, arachidonic acid, linoleic acid and linolenic acid.

63. The pharmaceutical composition of claim 54, wherein A is alkyl.

64. The pharmaceutical composition of claim 63, wherein each of X, Y, Z
and W is a carbon atom.

65. The pharmaceutical composition of claim 64, wherein each of D, R1, R2, R3 and R4 is hydrogen.

66. The pharmaceutical composition of claim 64, wherein D is alkyl and each of R1, R2, R3 and R4 is hydrogen.

67. The pharmaceutical composition of claim 63, wherein each of X, Y
and Z is a carbon atom and W is a nitrogen atom.

68. The pharmaceutical composition of claims 51-67, wherein at least one of R1, R2, R3 and R4 is a group containing at least one amino moiety.

69. The pharmaceutical composition of claim 68, wherein at least two of R1, R2, R3 anal R4 are said groups containing at least one amino moiety.

70. The pharmaceutical composition of claim 69, wherein each of R1 and R2 is said group containing at least one amino acid moiety.

71. The pharmaceutical composition of claim 69, wherein each of R3 and R4 is said group containing at least one amino acid moiety.

72. The pharmaceutical composition of claims 68-71, wherein said at least one amino moiety is selected from the group consisting of guanidino, guanidinoalkyl, amino, aminoalkyl, hydrazine, guanyl, guanylinoalkyl, and any combination thereof.

73. The pharmaceutical composition of claims 68-71, wherein said group containing at least one amino acid moiety comprises at least one positively charged group.

74. The pharmaceutical composition of claim 73, wherein said at least one positively charged group comprises an ammonium ion.

75. The pharmaceutical composition of claim 73, wherein said positively charged group has a chemical structure derived from a side chain of a positively charged amino acid.

76. The pharmaceutical composition of claim 75, wherein said positively charged amino acid is selected from the group consisting of arginine, lysine, histidine, proline and any derivative thereof.

77. The pharmaceutical composition of claim 51, packaged in a packaging material and identified in print, on or in said packaging material, for use in the treatment of a biological condition associated with GSK-3 activity.

78. The pharmaceutical composition of claim 77, wherein said biological condition is selected from the group consisting of obesity, non-insulin dependent diabetes mellitus, an insulin-dependent condition, an affective disorder, a neurodegenerative disease or disorder and a psychotic disease or disorder.

79. The pharmaceutical composition of claim 78, wherein said affective disorder is selected from the group consisting of a unipolar disorder and a bipolar disorder.

80. The pharmaceutical composition of claim 79, wherein said unipolar disorder is depression.

81. The pharmaceutical composition of claim 79, wherein said bipolar disorder is manic depression.

82. The pharmaceutical composition of claim 78, wherein said neurodegenerative disorder results from an event selected from the group consisting of cerebral ischemia, stroke, traumatic brain injury and bacterial infection.

83. The pharmaceutical composition of claim 78, wherein said neurodegenerative disorder is a chronic neurodegenerative disorder.

84. The pharmaceutical composition of claim 83, wherein said chronic neurodegenerative disorder 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.

85. The pharmaceutical composition of claim 51, further comprising at least one additional active ingredient that is capable of altering an activity of GSK-3.

86. The pharmaceutical composition of claim 85, wherein said additional active ingredient is capable of inhibiting an activity of GSK-3.

87. The pharmaceutical composition of claim 85, wherein said additional active ingredient is capable of downregulating an expression of GSK-3.

88. A method of inhibiting an activity of GSK-3, the method comprising contacting cells expressing GSK-3 with an inhibitory effective amount of a compound having a general formula:

wherein:
X, Y, Z and W are each independently a carbon atom or a nitrogen atom;
A is alkyl or absent;

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, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, cyano, nitro, 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 a hydrophobic moiety; and R1, 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, nitro, 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, said compound being capable of inhibiting an activity of GSK-3.

89. The method of claim 88, wherein A is alkyl.

90. The method of claim 88, wherein L is a phosphor atom.

91. The method of claim 90, wherein each of Q, G and D is oxygen.

92. The method of claim 91, wherein E is hydroxy.

93. The method of claim 88, wherein at least one of X, Y, Z and W is a nitrogen atom.

94. The method of claim 93, wherein at least two of X, Y, Z and W are nitrogen atoms.

95. The method of claim 94, wherein X and Y are each a nitrogen atom.

96. The method of claim 94, wherein Z and W are each a nitrogen atom.

97. The method of claim 88, wherein D is a hydrophobic moiety.

98. The method of claim 97, wherein said hydrophobic moiety is 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 40 carbon atoms, an aryl, a cycloalkyl and a hydrophobic peptide sequence.

99. The method of claim 98, wherein said fatty acid is selected from the group consisting of myristic acid, lauric acid, palmitic acid, stearic acid, oleic acid, arachidonic acid, linoleic acid and linolenic acid.

100. The method of claim 91, wherein A is alkyl.

101. The method of claim 100, wherein each of X, Y, Z and W is a carbon atom.

102. The method of claim 101, wherein each of D, R1, R2, R3 and R4 is hydrogen.

103. The method of claim 101, wherein D is alkyl and each of R1, R2, R3 and R4 is hydrogen.

104. The method of claim 100, wherein each of X, Y and Z is a carbon atom and W is a nitrogen atom.

105. The method of claims 88-104, wherein at least one of R1, R2, R3 and R4 is a group containing at least one amino moiety.

106. The method of claim 105, wherein at least two of R1, R2, R3 and R4 are said groups containing at least one amino moiety.

107. The method of claim 106, wherein each of R1 and R2 is said group containing at least one amino acid moiety.

108. The method of claim 106, wherein each of R3 and R4 is said group containing at least one amino acid moiety.

109. The method of claims 105-108, wherein said at least one amino moiety is selected from the group consisting of guanidino, guanidinoalkyl, amino, aminoalkyl, hydrazine, guanyl, guanylinoalkyl, and any combination thereof.

110. The method of claims 105-108, wherein said group containing at least one amino acid moiety comprises at least one positively charged group.

111. The method of claim 110, wherein said at least one positively charged group comprises an ammonium ion.

112. The method of claim 110, wherein said positively charged group has a chemical structure derived from a side chain of a positively charged amino acid.

113. The method of claim 112, wherein said positively charged amino acid is selected from the group consisting of arginine, lysine, histidine, proline and any derivative thereof.

114. The method of claim 88, wherein said activity is a phosphorylation activity and/or an autophosphorylation activity.

115. The method of claim 88, wherein said contacting is effected in vitro.

116. The method of claim 88, wherein said contacting is effected in vivo.

117. The method of claim 88, further comprising contacting said cells with at least one an additional active ingredient, said additional active ingredient being capable of altering an activity of GSK-3.

118. The method of claim 117, wherein said additional active ingredient is capable of inhibiting an activity of GSK-3.

119. The method of claim 117, wherein said additional active ingredient is capable of downregulating an expression of GSK-3.

120. A method of potentiating insulin signaling, the method comprising contacting insulin responsive cells with an effective amount of a compound having a general formula:

wherein:
X, Y, Z and W are each independently a carbon atom or a nitrogen atom;
A is alkyl or absent;

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, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, cyano, nitro, 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 a hydrophobic moiety; and R1, 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, nitro, 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, said compound being capable of inhibiting an activity of GSK-3.

121. The method of claim 120, wherein A is alkyl.

122. The method of claim 120, wherein L is a phosphor atom.

123. The method of claim 122, wherein each of Q, G and D is oxygen.

124. The method of claim 122, wherein E is hydroxy.

125. The method of claim 120, wherein at least one of X, Y, Z and W is a nitrogen atom.

126. The method of claim 125, wherein at least two of X, Y, Z and W are nitrogen atoms.

127. The method of claim 126, wherein X and Y are each a nitrogen atom.

128. The method of claim 126, wherein Z and W are each a nitrogen atom.

129. The method of claim 120, wherein D is a hydrophobic moiety.

130. The method of claim 129, wherein said hydrophobic moiety is 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.

131. The method of claim 130, wherein said fatty acid is selected from the group consisting of myristic acid, lauric acid, palmitic acid, stearic acid, oleic acid, arachidonic acid, linoleic acid and linolenic acid.

132. The method of claim 123, wherein A is alkyl.

133. The method of claim 132, wherein each of X, Y, Z and W is a carbon atom.

134. The method of claim 133, wherein each of D, R1, R2, R3 and R4 is hydrogen.

135. The method of claim 133, wherein D is alkyl and each of R1, R2, R3 and R4 is hydrogen.

136. The method of claim 132, wherein each of X, Y and Z is a carbon atom and W is a nitrogen atom.

137. The method of claims 120-136, wherein at least one of R1, R2, R3 and R4 is a group containing at least one amino moiety.

138. The method of claim 137, wherein at least two of R1, R2, R3 and R4 are said groups containing at least one amino moiety.

139. The method of claim 138, wherein each of R1 and R2 is said group containing at least one amino acid moiety.

140. The method of claim 138, wherein each of R3 and R4 is said group containing at least one amino acid moiety.

141. The method of claims 137-140, wherein said at least one amino moiety is selected from the group consisting of guanidino, guanidinoalkyl, amino, aminoalkyl, hydrazine, guanyl, guanylinoalkyl, and any combination thereof.

142. The method of claims 137-140, wherein said group containing at least one amino acid moiety comprises at least one positively charged group.

143. The method of claim 142, wherein said at least one positively charged group comprises an ammonium ion.

144. The method of claim 142, wherein said positively charged group has a chemical structure derived from a side chain of a positively charged amino acid.

145. The method of claim 144, wherein said positively charged amino acid is selected from the group consisting of arginine, lysine, histidine, proline and any derivative thereof.

146. The method of claim 120, further comprising contacting said cells with insulin.

147. The method of claim 120, wherein said contacting is effected in vitro.

148. The method of claim 120, wherein said contacting is effected in vivo.

149. A method of treating a biological condition associated with an activity of GSK-3, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound having a general formula:

wherein:

X, Y, Z and W are each independently a carbon atom or a nitrogen atom;
A is alkyl or absent;

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, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, cyano, nitro, 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, hydrazine and a hydrophobic moiety;
and R1, 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, nitro, 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, said compound being capable of inhibiting an activity of GSK-3.

150. The method of claim 149, wherein A is alkyl.

151. The method of claim 149, wherein L is a phosphor atom.

152. The method of claim. 151, wherein each of Q, G and D is oxygen.

153. The method of claim 152, wherein B is hydroxy.

154. The method of claim 149, wherein at least one of X, Y, Z and W is a nitrogen atom.

155. The method of claim 154, wherein at least two of X, Y, Z and W are nitrogen atoms.

156. The method of claim 155, wherein X and Y are each a nitrogen atop.

157. The method of claim 155, wherein Z and W are each a nitrogen atom.

158. The method of claim 149, wherein D is a hydrophobic moiety.

159. The method of claim 158, wherein said hydrophobic moiety is 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.

160. The method of claim 159, wherein said fatty acid is selected from the group consisting of myristic acid, lauric acid, palmitic acid, stearic acid, oleic acid, arachidonic acid, linoleic acid and linolenic acid.

161. The method of claim 152, wherein A is alkyl.

162. The method of claim 161, wherein each of X, Y, Z and W is a carbon atom.

163. The method of claim 162, wherein each of D, R1, R2, R3 and R4 is hydrogen.

164. The method of claim 162, wherein D is alkyl and each of R1, R2, R3 and R4 is hydrogen.

165. The method of claim 161, wherein each of X, Y and Z is a carbon atom and W is a nitrogen atom.

166. The method of claims 149-165, wherein at least one of R1, R2, R3 and R4, is a group containing at least one amino moiety.

167. The method of claim 166, wherein at least two of R1, R2, R3 and R4 are said groups containing at least one amino moiety.

168. The method of claim 167, wherein each of R1 and R2 is said group containing at least one amino acid moiety.

169. The method of claim 167, wherein each of R3 and R4 is said group containing at least one amino acid moiety.

170. The method of claims 16d-169, wherein said at least one amino moiety is selected from the group consisting of guanidino, guanidinoalkyl, amino, aminoalkyl, hydrazine, guanyl, guanylinoalkyl, and any combination thereof.

171. The method of claims 166-169, wherein said group containing at least one amino acid moiety comprises at least one positively charged group.

172. The method of claim 171, wherein said at least one positively charged group comprises an ammonium ion.

173. The method of claim 171, wherein said positively charged group has a chemical structure derived from a side chain of a positively charged amino acid.

174. The method of claim 173, wherein said positively charged amino acid is selected from the group consisting of arginine, lysine, histidine, proline and any derivative thereof.

175. The method of claim 149, wherein said biological condition is selected from the group consisting of obesity, non-insulin dependent diabetes mellitus, an insulin-dependent condition, an affective disorder, a neurodegenerative disease or disorder and a psychotic disease or disorder.

176. The method of claim 175, wherein said affective disorder is selected from the group consisting of a unipolar disorder and a bipolar disorder.

177. The method of claim 176, wherein said unipolar disorder is depression.

178. The method of claim 176, wherein said bipolar disorder is manic depression.

179. The method of claim 175, wherein said neurodegenerative disorder results from an event selected from the group consisting of cerebral ischemia, stroke, traumatic brain injury and bacterial infection.

180. The method of claim 175, wherein said neurodegenerative disorder is a chronic neurodegenerative disorder.

181. The method of claim 180, wherein said chronic neurodegenerative disorder 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.

182. The method of claim 175, wherein said psychotic disorder is schizophrenia.

183. The method of claim 149, further comprising co-administering to said subject at least one additional active ingredient, said at least one additional active ingredient being capable of altering an activity of GSK-3.

184. The method of claim 183, wherein said additional active ingredient is capable of inhibiting an activity of GSK-3.

185. The method of claim 183, wherein said additional active ingredient is capable of downregulating an expression of GSK-3.