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Definitions

• 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.
• GSK-3 glycogen synthase kinase-3
• 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.
• Glycogen synthase kinase-3 (GSK-3), a member of the protein kinases family, is a cytoplasmic proline-directed serme-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.
• GSK-3 and GSK-3 ⁇ Two similar isofor s of the enzyme, termed GSK-3 and GSK-3 ⁇ , 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.
• PDB protein kinase B
• Akt protein kinase B
• the intricate insulin-signaling pathway is further complicated by negative-feedback regulation of insulin signaling by GSK-3 itself, which phosphorylates insulin-receptor substrate- 1 on serine residues (Eldar- Finkelman et al., 1997). 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.
• glycogen synthase GS
• TRS-1 insulin receptor substrates
• IRS-2 insulin receptor substrates
• PI3 kinase Myers et al, 1992
• the activity of glycogen synthase is suppressed by its phosphorylation.
• glycogen synthase activity 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).
• 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.
• GSK-3 is also considered to be an important player in the pathogenesis of
• 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).
• GSK-3 is further linked with Alzheimer's disease by its role in cell apoptosis.
• GSK-3 Activation of GSK-3 in cerebellar granule neurons mediated migration and cell death (Tong et al., 2001).
• SH-SY5Y cells over expression of GSK-3 facilitated stauroaporine-induced cell apoptosis (Bijur et al., 2000).
• Fratl a GSK-3 ⁇ inhibitor
• Another implication of GSK-3 was detected in the context of affective disorders, i.e., bipolar disorders and manic depression.
• 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).
• AML amyotrophic lateral sclerosis
• MS multiple sclerosis
• GSK-3 inhibitors are believed to be a useful treatment in these and other neurodegenerative disorders.
• dysregulation of GSK-3 activity has been recently implicated in several C ⁇ S disorders and neurodegenerative diseases, including schizophrenia (Beasley et al., 2001; Kozlovsky et al., 2002), stroke, and Alzheimer's disease (AD) (Bhat and Budd, 2002; Hernandez et al., 2002; Lucas et al., 2001 ; Mandelkow et al., 1992).
• 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).
• 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.
• GSK-3 inhibitors 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.
• CDK-2 cyclic-dependent protein kinase-2
• 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.
• 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
• 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, sulf ⁇ nyl, sulfonyl, cyano, nitro, azo,
• GSK-3 is alkyl.
• L is a phosphor atom.
• each of Q, G and D is oxygen, and E is preferably hydroxy.
• at least one of X, Y, Z and W is a nitrogen atom.
• at least two of X, Y, Z and W are nitrogen atoms.
• X and Y are each a nitrogen atom or Z and W are each a nitrogen atom.
• R 2 , R 3 and R4 is a group containing at least one amino moiety.
• at least two of R 1 ⁇ R 2 , R 3 and R 4 are groups containing at least one amino moiety.
• each of R t and R or each of R 3 and is a group containing at least one amino moiety.
• groups containing at least one amino moiety include, without limitation, guanidino, guanidinoalkyl, aminoalkyl, analogs thereof, derivatives thereof and any combination thereof.
• the group containing at least one amino moiety comprises at least one positively charged group
• the positively charged group comprises an ammonium ion.
• 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, argi ine, lysine, histidine, proline and any derivative thereof.
• 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 Ri,
• R 2 , R 3 and R 4 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.
• 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 carrier.
• 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.
• 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.
• 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 amount of a compound that is capable of inhibiting an activity of GSK-3, as is described hereinabove.
• 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 or 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 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.
• a 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.
• a method of potentiating insulin signaling which comprises contacting insulin responsive cells with an effective amount of the compound described hereinabove.
• the contacting the cells can be effected in vitro or in vivo.
• 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.
• 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;
• FIGs. 4a-b present the ! H NMR spectrum ( Figure 4a) and the 13 C NMR spectrum ( Figure 4b) of l,3,5-tris(hydroxylmethyl)benzene, an intermediate in the synthesis of GS-21;
• FIGs. 5a-b present the ! H NMR spectrum ( Figure 5a) and the 13 C NMR spectrum ( Figure 5b) of 3,5-Bis(bromomethyl)benzyl alcohol, an intermediate in the synthesis of GS-21;
• FIG. 6a-b present the 1H NMR spectrum ( Figure 6a) and the 13 C NMR spectrum ( Figure 6b) of 3,5-Bis(cyanomethyl)benzyl alcohol, an intermediate in the synthesis of GS-21;
• FIG. 7 presents the 1H NMR spectrum of 3,5-bis(aminoethyl)benzyl alcohol, an intermediate in the synthesis of GS-21 ;
• FIG. 8 presents the 1H NMR spectrum of 3,5-bis(tert- butoxycarbonylaminoethyl)benzyl alcohol, an intermediate in the synthesis of GS-21;
• FIGs. 9a-c present the 1H NMR spectrum ( Figure 9a) and the 13 C NMR spectrum ( Figure 9b) and the 31 P NMR spectrum ( Figure 9c) of protected 3,5-Bis(2- aminoethyl)benzyl phosphate, an intermediate in the synthesis of GS-21 ;
• FIGs. lOa-d present the 1H NMR spectrum ( Figure 10a) and the 13 C NMR spectrum ( Figure 10b), the 31 P NMR spectrum ( Figure 10c) and the ESI-MS ( Figure lOd) of the TFA salt 3,5-Bis(2-aminoethyl)benzyl phosphate (GS-21 TFA salt); FIGs.
• 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-1, GS-2, GS-3, GS-5 and GS-21 in in vitro inhibition assays
• 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 [ 3 H] 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).
• 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.
• 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.
• GSK-3 unlike other kinases, has a unique recognition motif, which includes the amino acid sequence SXiX 2 X 3 S(p), set forth in SEQ ID NO:l, where S is serine or threonine, each of Xi, X 2 and X 3 is any amino acid, and S(p) is phosphoiylated serine or phosphorylated threonine.
• S(p) is phosphoiylated serine or phosphorylated threonine.
• GSK-3 recognizes only pre-phosphorylated substrates, namely, substrates that have a phosphorylated serine or threonine residue. 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 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.
• a GSK-3 substrate the short pre- phosphorylated peptide p9CREB (ILSRRPS( ⁇ )YR, SEQ ID NO:2) was selected.
• Figure 2 presents the electrostatic distribution on the 'surface' of the p9CREB peptide, based on these findings.
• the molecule should include a negatively charged group, preferably a phosphate group;
• 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.
• GSK-3 activity has been designed. As is described in the Examples section that 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 inhibitory potential of compounds having such a formula. 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, 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 inhibitory potential of compounds having such a formula. A more advanced generation of compounds, which includes novel compounds, was also designed and synthesized based on the above. As is described in the
• 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 tb-e 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;
• 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, thio
• alkyl refers to a saturated aliphatic hydrocarbon including straight chain and branched chain groups.
• the alkyl group has 1 to 20 carbon atoms.
• 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.
• the substituent group can be, for example, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, cyano, nitro, 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,
• 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.
• the substituent group can be, for example, alkyl, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, cyano, nitro, 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-ca boxy,
• alkenyl refers to an alkyl group, as defined hereinabove, which consists of at least two carbon atoms and at least one carbon-carbon double bond.
• alkynyl refers to an alkyl group, as defined hereinabove, which consists of at least two carbon atoms and at least one carbon-carbon triple bond.
• aryl refers to an all-carbon monocyclic or fused-ring polycyclic (i.e., 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.
• the substituent group can be, for example, alkyl, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, cyano, nitro, 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,
• heteroaryl refers to a monocyclic or fused ring (i.e., rings which share an adjacent pair of atoms) group having in the ring(s) one or more atoms, such as, for example, nitrogen, oxygen and sulfur and, in addition, having a completely conjugated pi-electron system.
• heteroaryl groups examples include pyrrole, furane, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline and purine.
• the heteroaryl group may be substituted or unsubstituted.
• the substituent group can be, for example, alkyl, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, cyano, nitro, 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, O-
• 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 unsubstituted.
• the substituted group can be, for example, lone pair electrons, alkyl, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, cyano, nitro, 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-carbox
• 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 “alkoxy” group refers to both an -O-alkyl and an -O-cycloalkyl group, as defined herein.
• aryloxy 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.
• An "aldehyde” group refers to a carbonyl group, where R' is hydrogen.
• 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 -CX 3 group wherein X is a halo group as defined herein.
• R are as defined herein.
• An “amino” group refers to an -NR'R” group where R' and R" are as defined herein.
• aminoalkyl refers to an alkyl, as defined hereinabove, substituted by an amino group. Preferably, the alkyl terminates by the amino group.
• R" and R" ' are as defined herein and R" " is defined as either R' , R" or R' " .
• a “guanidinoalkyl” group refers to an alkyl group substituted by a guanidino group, as these terms are defined herein. Preferably, the alkyl group terminates by the guanidino group.
• a “guanylinoalkyl” group refers to an alkyl group substituted by a guanyl group, as these terms are defined herein. Preferably, the alkyl group terminates by the guanyl group.
• a "nitro” group refers to a -NO 2 group.
• a “cyano” group refers to a -C ⁇ N group.
• the term “hydrazine” describes a NR'-NR' ' group, with R' and R' ' as defined hereinabove.
• the term “ammonium ion” described a (NR'R"R') + , 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.
• 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.
• 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.
• 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.).
• 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.
• E is hydroxy.
• the hydroxy group can also be ionized so as to have another negative electrostatic charge.
• the negatively charged group can be a thiophosphate group, sulfate or sulfonate group, a borate or boronate group and the like, according to the 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.
• a in the formula above is alkyl, preferably an unsubstituted alkyl, and more preferably a methyl.
• 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.
• 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).
• the compounds according to this aspect of the invention exclude any of the presently known compounds that are embraced by the formula above.
• the base structure of the compounds of the present invention is an aromatic ring or a heteroaromatic ring.
• 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.
• Z or W is a nitrogen atom.
• 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.
• a compound that has one or two of such positively charged groups flanking the negatively charged group is preferable.
• Ri, R 2 , R 3 and R 4 is a group containing at least one amino moiety.
• 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.
• 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.
• 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 -NH 3 + group, for example.
• a compound that has one or two of such positively charged groups flanking the negatively charged group is preferable.
• 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.
• 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.
• 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.
• R ! and R 2 or R 3 and R 4 are groups containing at least one amino moiety (e.g., positively charged groups), which flank the negatively charged group, as desired.
• preferred compounds according to the present invention are those having the following formulas:
• m is an integer from 1 to 6; each of Q t and Q 2 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).
• G and/or K are each a group containing a free amino moiety (e.g., a positively charged group).
• Z and W is a carbon atom; and at least one of R 3 and R 4 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.
• 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.
• hydrophobic moiety refers to any substance or a residue thereof that is characterized by hydrophobicity.
• 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 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.
• 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 further 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.
• the hydrophobic moiety, according to the present invention can be a hydrophobic peptide sequence.
• the hydrophobic peptide sequence preferably includes between 2 and 15 amino acid residues, more preferably between 2 and 10 amino acid residues, more preferably between 2 and 5 amino acid residues, in which at least one amino acid residue is a hydrophobic amino acid residue.
• 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.
• the hydrophobic amino acid residue can include any other amino acid residue, which has been modified by incorporation of a hydrophobic moiety thereto.
• 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.
• 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.
• 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.
• 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 peniciUamine, 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.
• 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, phosphoserine and phosphothreonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor- leucine and orrnti-ine.
• 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.
• 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.
• the compounds of the present invention, described hereinabove are designed based on the tl-iree-dimensional structure of a GSK-3 substrate and are therefore potential substrate competitive inhibitors of GSK-3 activity.
• 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.
• the te ⁇ n "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 in 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-Finkehnan et al, 1997). This impairment results from the phosphorylation of insulin receptor substrate-1 (IRS-1) serine residues by GSK-3.
• IRS-1 insulin receptor substrate-1
• NDDDM non-insulin dependent diabetes mellitus
• 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.
• 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.
• 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, in vitro or in vivo, and can be also effected by further contacting the cells with insulin.
• Potentiation of insulin signaling, in vivo, resulting from administration of the conjugates of the present invention can be monitored as a clinical endpoint.
• 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.
• a method of treating a biological condition associated with GSK-3 activity is provided.
• the method is effected by administering to a subject in need thereof a therapeutically effective amount of the compound of the present invention, described hereinabove.
• biological condition associated with GSK-3 activity 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 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.
• 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 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.
• administering describes a method for bringing the 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, 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 also be intra-articularly, intrarectally, intraperitoneally, intramuscularly, subcutaneously, or by aerosol inhalant.
• 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.
• 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.
• 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
• 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.
• 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.
• GSK-3 inhibitors are known to inhibit differentiation of pre-adipocytes into adipocytes
• the method of this aspect of the present invention can be used to treat obesity.
• 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 ⁇ 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.
• NIDDM Type II diabetes mellitus
• 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 (mg/dl) before meals and 100-140 mg/dl 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 dete ⁇ nine blood sugar level, tests for glycated hemoglobin level (HbA lo ; a measure of 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.
• 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
• 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).
• the method according to this aspect of the present invention can be further used to treat a variety of such disorders and diseases.
• 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.
• 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, AIDS associated dementia, amyotrophic lateral sclerosis (AML) and multiple sclerosis.
• GSK-3 activity has particularly been implicated in the pathogenesis of Alzheimer's disease.
• 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).
• BBB blood brain barrier
• the patient is monitored for tau phosphorylated polymers by periodic analysis of proteins isolated from the patient's 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) which is capable of altering an activity of GSK-3.
• 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, 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.
• the additional active ingredient can be insulin.
• 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 U.S. Patent Application Publication No. 20020147146A1.
• the GSK-3 inhibitor can be, for example, lithium, valproic acid and/or lithium ion.
• 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 affects GSK-3 synthesis (decelerates) or degradation (accelerates) either at the level of the mRNA or at the level of the protein.
• a small interfering polynucleotide molecule which is designed to down regulate the expression of GSK-3 can be used as an additional active ingredient according to this embodiment of the present invention.
• 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.
• 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.
• 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.
• dsRNA double-stranded RNA
• ssRNA single-stranded RNA
• isolated RNA i.e., partially purified RNA, essentially pure RNA
• synthetic RNA recombinantly produced RNA.
• 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 www.atnbion .com.
• the small interfering polynucleotide molecule according to the present invention can be an RNAi molecule (RNA interference molecule).
• a small interfering polynucleotide molecule can be an oligonucleotide such as a GSK-3-s ⁇ ecific 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.
• 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, and/or increased binding affinity for the target polynucleotide.
• An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving R A: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.
• RNA target 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. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065;
• 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.
• sequences include but are not limited ANGIOZYME specifically inhibiting formation of the NEGF-R (Vascular Endothelial Growth Factor receptor), a key component in the angiogenesis pathway, and HEPTAZYME, a rybozyme designed to selectively destroy Hepatitis C Nirus (HCN) R ⁇ A, (Rybozyme Pharmaceuticals, Incorporated - WEB home page).
• a small interfering polynucleotide molecule can be a D ⁇ Azyme.
• D ⁇ Azymes are single-stranded catalytic nucleic acid molecules. A general model (the "10-23" model) for the D ⁇ Azyme has been proposed.
• D ⁇ Azymes have a catalytic domain of 15 deoxyribonucleotides, flanked by two substrate-recognition domains of seven to nine deoxyribonucleotides each.
• This type of D ⁇ Azyme can effectively cleave its substrate R ⁇ A at purme yrimidine junctions (Santoro, S.W. & Joyce, G.F. Proc. ⁇ atl, Acad. Sci. USA 199; for rev of D ⁇ Azymes see Khachigian, LM Curr Opin Mol Ther 2002;4: 119-21). Examples of construction and amplification of synthetic, engineered D ⁇ Azymes recognizing single and double-stranded target cleavage sites have been disclosed in U.S.
• 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 executing solid-phase synthesis are commercially available from, for example,
• 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.
• a pharmaceutical composition which comprises, as an active ingredient, a compound according to the present invention and a pharmaceutically acceptable carrier.
• the phrases “pharmaceutically acceptable carrier” and “physiologically acceptable carrier” 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.
• carriers are propylene glycol, saline, emulsions and mixtures of organic solvents with water.
• excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound.
• excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
• the pharmaceutical acceptable carrier can further include other agents such as, but not limited to, absorption delaying agents, antibacterial agents, antifungal agents, antioxidant agents, binding agents, buffering agents, bulking agents, cationic lipid agents, coloring agents, diluents, disintegrants, dispersion agents, emulsifying agents, excipients, flavoring agents, glidants, isotonic agents, Uposomes, microcapsules, solvents, sweetening agents, viscosity modifying agents, wetting agents, and skin penetration enhancers. Techniques for formulation and administration of drugs may be found in
• 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 lyophiUzing processes.
• 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.
• 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.
• 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.
• organic solvents such as propylene glycol, polyethylene glycol.
• penetrants are used in the formulation. Such penetrants are generally known in the art.
• 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 cellulose, hydroxypropy-methyl-cellulose, sodium carbomethylceUulose and/or physiologicaUy acceptable polymers such as polyvinylpyrroUdone (PNP).
• PNP polyvinylpyrroUdone
• 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.
• suitable coatings 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.
• 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.
• the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
• stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
• compositions may take the form of tablets or lozenges formulated in conventional manner.
• 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, tiichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
• a suitable propellant e.g., dichlorodifluoromethane, tiichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
• 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.
• 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 Uposomes. 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.
• the compound may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.
• a suitable vehicle e.g., sterile, pyrogen-free water
• 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.
• 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 therapeuticaUy 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.
• 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.
• 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 bUster 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 Usted hereinabove.
• 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 GSK-3.
• 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., Sigma, Acres, Aldrich) and were used as supplied, unless otherwise indicated.
• GS-1, 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.
• 2D 1H NMR spectroscopy was determined using the following procedures: For the structural studies, a solution of each peptide was prepared by dissolving lyophUized powder in water containing 10 % D 2 O. 2D-NMR spectra were acquired at the 1H proton frequency of 600.13 MHz on a Bruker 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.
• the data was processed using Bruker XWINNMR software (Bruker Analytician Messtechnik, GmbH, version 2.1). AU data processing, calculations and analysis were done on Silicon Graphics workstations (INDY R4000 and INDIGO2 R10000). 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 Bruker. Resonance assignment was based on the TOCSY and NOESY spectra measured at the same experimental conditions, according to the sequential assignment methodology developed by Wuthrich using the Bruker software program AURELIA
• 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 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 f 6 dependency and the known distance of 2.47 A between the two adjacent protons of the tyrosine aromatic ring was used for calibration. The restraints were classified into strong (1.8-2.5 A), medium (1.8-3.5 A) and weak (1.8-5.0 A).
• TLC Thin-layer chromatography
• UV ultraviolet
• 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:
• 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 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.
• the mixture was cooled to -40 °C (by means of dry ice/acetonitrile), and a solution of 85 % m-chloroperbenzoic acid (mCPBA) (0.81 gram in 1 ml dichloromethane, 4 mmol, 1.3 equivalents) in dichloromethan (4 mL) was rapidly added while the reaction temperature was kept below 0 °C.
• mCPBA m-chloroperbenzoic acid
• the solution was allowed to warm up to room temperature and after stirring for 5 minutes at 20 °C, 10 % aqueous NaHSO 3 (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 NaHSO 3 (2 x 20 ml), 5 % saturated aqueous NaHCO 3 (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.
• the ethereal phase was washed with 10 % aqueous NaHSO 3 (2 x 20 ml), 5 % saturated aqueous NaHCO 3 (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 CHCl 3 /hexanes 1 : 1 as eluent, to give a mixture of di-tert-butyl, 3-Pyridylmethyl phosphate and the starting material, which was used without further purification.
• 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 5.
• 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 w-chloroperbenzoic acid (mCPBA) yielded the corresponding phosphate ester.
• mCPBA w-chloroperbenzoic acid
• 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 depicted in Scheme 6 below.
• the reaction mixture then was washed successively with 1.0 M aqueous solution of sodium thiosulfate (100 ml) and saturated sodium bicarbonate (2 x 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 yeUow oil, which was then purified by column chromatography (sUica gel, 0-5 % MeOH/CH 2 Cl 2 ).
• the protected 3,5-Bis(2- aminoethyl)benzyl phosphate (2.1 grams, 61 %) was obtained as a viscous, colorless oil.
• Di-tert-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 t der 6 °C.
• the crude material was purified by column chromatography (silica gel,
• the ethereal phase was washed with 10 % aqueous NaHSO 3 (2 x 20 ml) and saturated aqueous NaHCO 3 (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 CHCl 3 :MeOH 30:1 to 20:1), to give pure di-tert-butyl, 3-(N,N'-bis-BOC- guanidinomethy) benzyl phosphate in 70 % yield.
• results, presented in Figure 13 represent the percentage of GSK-3 activity as compared with a control incubation without inhibitors and are mean of 2 independent experiments ⁇ SEM, where each point was assayed in triplicate. As is shown in Figure 13, all the tested compounds were found highly active in inhibiting GSK-3 activity (IC50 values of 1-5 mM), with GS-3 and GS-5 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-5 and GS-21 to promote glucose uptake was tested in mouse primary adipocytes as described hereinabove. The relative [ 3 H] 2-deoxy glucose incorporation observed in non-treated adipocytes was normalized to 1 unit and the values obtained for [ 3 H] 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 ⁇
• ATOM 13 HD11 ILE 1 10.871 -3.209 -3.300 1.00 0.00
• ATOM 110 HD2 PRO 6 -3.044 3, .093 0. .948 1. .00 0. .00
• ATOM 134 HD1 TYR 8 -6.664 4 .205 0 .901 1. .00 0, .00
• ATOM 136 HD2 TYR 8 10.261 2. .331 2. .352 1. .00 0. .00
• ATOM 148 CA ARG 9 11.423 -1. .283 -1. .334 1. .00 0. .00
• ATOM 157 HD1 ARG 9 11.932 -4. .342 -1. .351 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 1.00 0.00
• ATOM 103 CB PRO 6 2. .877 5.579 0, .665 1. .00 0, .00
• ATOM 110 HD2 PRO 6 1. .038 2.890 0, .343 1. .00 0. ,00
• ATOM 111 HD1 PRO 6 0 ' . .316 4.415 -0. .219 1. .00 0. ,00
• ATOM 112 C PRO 6 4. .587 4.040 -0, .334 1. .00 0. .00
• ATOM 115 HN SRP 7 4. .501 3.180 -2. .153 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 0G4 SRP 7 9. .056 6.362 -1. .210 1. ,00 0, ,00
• ATOM 128 HB1 SRP 7 8. .290 2.553 -0 .751 1, .00 0. .00
• ATOM 140 CD2 TYR 8 1, .202 1.269 2. .544 1. .00 0. .00
• ATOM 161 CD ARG 9 10. .085 -2.996 0 .895 1, .00 0. .00 72
• 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. . ⁇ O 0. .00
• J Glycogen synthase kinase-3 beta facilitates staurosporine- and heat shock-induced apoptosis. Protection by lithium J Biol Chem 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 Comm
• Hsp70 protein in Saccharomyces cerevisiae 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 locaUsation of the kinase.
• Hawiger J "Cellular import of functional peptides to block intracellular signaling Curr Opin Immunol 9(2):189-194 (1997) Hawiger, J. Curr. Opin. Immun. 9, 189-194 (1997) Hawiger, J.
• Kole et al "Protein-tyrosine phosphatase inhibition by a peptide containing the phosphotyrosyl mimetic, L-O-malonyltyrosine", Biochem 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", Indian J Biochem Biophys 34(l-2):50-55 (1997) Latimer et al, "Stimulation of MAP kinase by v-raf transformation of fibroblasts fails to induce hyperphosphorylation of transfected tau", EERS Lett

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